Herpes simplex virus US3 and ICP4 as inhibitors of apoptosis

ABSTRACT

The ICP4 protein of herpes simplex virus plays an important role in the transactivation of viral genes. The present invention discloses that ICP4 also has the ability to inhibit apoptosis. This function appears to reside in functional domain distinct from the transactivating function, as indicated by studies using temperature sensitive mutants of ICP4 that transactivating function at elevated temperatures. Also disclosed are methods for inhibition of apoptosis using ICP4 or an ICP4 encoding gene, such as an α4 gene, methods of inhibiting ICP4&#39;s apoptosis-inhibiting function, and methods for the production of recombinant proteins and treatment of HSV infections. Further, the present invention discloses that the HSV-1 mutant lacking the α4 gene, has a secondary mutation in the gene U s 3 specifying a protein kinase. Thus a functional U s 3, a viral gene encoding a protein kinase known to phosphorylate serine/theonine within a specific arginine rich consensus sequence, is required in order to block apoptosis. Also disclosed are methods for inhibition of apoptosis using U s 3 or an U s 3 encoding gene, methods of inhibiting U s 3&#39;s apoptosis-inhibiting function and methods for the production of recombinant proteins and treatment of HSV infections.

BACKGROUND OF THE INVENTION

The government may own certain rights in this application by virtue offederal funding under grant numbers AI124009 (NIAID) and CA47451 (NCI).

I. Field of the Invention

The present invention relates to the fields of molecular and cellbiology generally, and more specifically, it addresses mechanisms forgrowth control in eurkaryotic cells. In particular, there are providedviral genes that inhibit normal cell death and methods for use thereof.

II. Related Art

The control of host cell gene expression, and often the control of genesinvolved in DNA replication, are integral parts of the life cycle of avirus. However, recent evidence suggests that most eukaryotic cellsrespond to viral disruption of normal cellular physiology by undergoingprogrammed cell death (apoptosis) (White, 1993). To counteract this,many viruses have evolved mechanisms to block host cell death (Clem andMiller, 1994; White and Gooding, 1994). In several cases, viral genomeshave been found to contain genes whose products interact with proteinsthat play a central role in regulating cell survival.

Programmed cell death is triggered by several factors and may takevarious forms. For example, the synthesis of double-stranded RNAactivates kinases which phosphorylate the α subunit of eIF-2 andcompletely turn off protein synthesis (Sarre, 1989). Ultimately,activation of metabolic pathways causes a pattern of morphological,biochemical, and molecular changes which result in cell death withoutspillage of cellular constituents which would result in an inflammatoryresponse detrimental to the host (Wyllie, et al.).

Apoptotic cell death is commonly observed during embryogenesis and organinvolution and in the natural death of terminally differentiated cellsat the end of their life span. Most viruses which induce either theshut-off of protein synthesis or apoptosis also have evolved mechanismswhich block host responses and enable them to replicate in their hosts(Shen and Shenk, 1995). Among the best-known examples of viral geneproducts which block apoptosis are the adenovirus E1B M_(r)19,000protein (Rao, et al, 1992.), vaccinia CmrA protein (Ray, et al.), simianvirus 40 (SV40) T antigen (McCarthy, et al., 1994), human papillomavirusNo. 16 (HPV 16) E6 protein (Pan and Griep, 1994), Epstein-Barr virusBHRF1 protein (Henderson, et al., 1993) and human cytomegalovirus IE1and IE2 gene products (Zhu, et al., 1995). Herpes simplex virus 1(HSV-1) encodes a protein, λ₁34.5, which blocks the phosphorylation ofeIF-2α (Chou and Roizman, 1992).

The utility of proteins that are capable of inhibiting apoptosis aremanifold. First, such proteins, or their corresponding genes, may beused to immortalize cell lines that otherwise would perish duringculture. This makes possible not only the study of these cells, but alsopresents the option of growing these cells in large numbers in order toisolate protein species therefrom. Second, the identification ofinhibitors of apoptosis and their function permits the possibleintervention, in a clinical setting, when these proteins are interferingwith normal programmed cell death, or apoptosis. This may beaccomplished by providing an inhibitor or an antisense nucleic acid thatinterferes with the expression of a protein that interferes withapoptosis. Thus, the identification of novel proteins having theseactivities and uses provide important new tools for those working inthis arena.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide methodsfor the use of Us₃ and its cognate gene Us₃ as inhibitors of apoptosis.The present invention further provides infected cell protein number 4,or ICP4, and its cognate gene α4, as inhibitors of apoptosis. Inaddition, it is an object of the present invention to provide methodsfor the use of agents that inhibit US3 protein function and geneexpression to induce apoptosis in HSV infected cells. The presentinvention also provides methods for the use of agents that inhibit theapoptosis-induction function of US3 thereby to inhibiting apoptosis invirally infected cells. It is a further objective to provide methods ofuse of agents that inhibit ICP4 and/or α4 in order to induce apoptosisin HSV infected cells. It also is an object of the present invention toprovide methods for the identification of agents that inhibit theapoptosis-inhibiting function of ICP4.

In satisfying these goals, there is provided a method for blockingapoptosis of a cell comprising the step of providing to the cell an HSVUs₃ polypeptide or an HSV Us₃ gene. The U_(s)3 gene may be contained inan expression vector and, further, under the control of a promoteractive in eukaryotic cells. One such promoter is the a tetracyclinecontrolled promoter. The expression vector further comprises aselectable marker and/or further comprises a gene encoding a secondpolypeptide under the transcriptional control of a promoter active ineukaryotic cells.

The method may further comprise the step of providing to the cell an HSVICP4 polypeptide or an HSV α4 gene. The α4 gene may be contained in anexpression vector and, further, under the control of a promoter activein eukaryotic cells.

In another embodiment, there is provided a method for inducing apoptosisin a cell infected with HSV comprising the step of administering to saidcell an agent that inhibits HSV U_(s)3 function in said cell. The agentmay inhibit transcription or translation of an HSV U_(s)3 gene ortranscript or may bind to an HSV U_(s)3 polypeptide. The reagent may bean antisense HSV U_(s)3 construct an antibody that binds immunologicallyto an HSV U_(s)3 polypeptide. Particular antisense constructs areoligonucleotides that hybridizes to a 5′-untranslated region for an HSVU_(s)3 gene or a translational start site for an HSV U_(s)3 transcript.Particular antibodies are polyclonal sera against U_(s)3 or a monoclonalantibody against U_(s)3.

In another embodiment, the method further comprising the step ofadministering to said cell an agent that inhibits HSV ICP4 function insaid cell.. The agent may inhibit transcription or translation of an HSVα4 gene or transcript or may bind to an HSV ICP4 polypeptide. Thereagent may be an antisense HSV α4 construct an antibody that bindsimmunologically to an HSV ICP4 polypeptide. Particular antisenseconstructs are oligonucleotides that hybridizes to a 5′-untranslatedregion for an HSV α4 gene or a translational start site for an HSV α4transcript. Particular antibodies are polyclonal sera against ICP4 or amonoclonal antibody against ICP4. The agent that inhibits ICP4 functionmay be administered prior to, concurrently with or after theadministration of the agent that inhibits U_(s)3 function.

In yet another embodiment, there is provided a method for treating asubject with an HSV infection comprising the step of inhibiting HSV US3function. The inhibition may comprise providing to the subject a firstpharmaceutical composition comprising an HSV U_(s)3 antisense constructor a monoclonal antibody that binds immunologically to an HSV U_(s)3polypeptide. The first pharmaceutical composition may be appliedtopically to HSV infected cells in said patient. The method may furthercomprise the step of providing to said subject a second pharmaceuticalcomposition comprising a conventional anti-HSV agent, such as acyclovir.Acyclovir is delivered via a route selected from the group consisting oftopically, orally and intravenously.

In a further aspect of the present invention the method for treating asubject with an HSV infection may further comprise the step ofinhibiting HSV ICP4 function. The inhibition may comprise providing tothe subject a first pharmaceutical composition comprising an HSV α4antisense construct or a monoclonal antibody that binds immunologicallyto an HSV ICP4 polypeptide. The first pharmaceutical composition may beapplied topically to HSV infected cells in said patient. The method mayfurther comprise the step of providing to said subject a secondpharmaceutical composition, comprising a conventional anti-HSV agent,such as acyclovir. Acyclovir is delivered via a route selected from thegroup consisting of topically, orally and intravenously.

In yet still another embodiment, there is provided a screening methodfor compounds having inhibitory activity against HSV U_(s)3polypeptide-induced inhibition of apoptosis comprising the steps of (a)providing a first cell comprising an HSV US3 gene under the control ofan HSV immediate early promoter; (b) infecting said first cell with aherpes simplex virus that lacks a functional U_(s)3 gene; (c) contactingsaid first cell with a test compound; (d) incubating said first cellunder conditions permitting viral replication; and (e) comparing thecell pathology of said first cell following incubation with the cellpathology of a second cell that lacks an HSV U_(s)3 gene followinginfection with said herpes simplex virus and the cell pathology of athird cell comprising an HSV U_(s)3 gene under the control of an HSVimmediate early promoter following infection with said herpes simplexvirus but in the absence of said test compound. Cell pathology comprisescondensation of chromatin, obliteration of nuclear membranes,vacuolization, cytoplasmic blebbing and DNA fragmentation.

The screening method may employ a cell line which contains an integratedcopy of a wild-type HSV U_(s)3 gene under the control of an α4 promoterand a herpes simplex virus that lacks a functional HSV U_(s)3 gene has adeletion in both copies of the virally-encoded U_(s)3 genes. Forexample, the herpes virus may carry a temperature sensitive mutation inboth copies of the virally-encoded U_(s)3 gene; incubation is at 39.5°C.

Another screening method for compounds having inhibitory activityagainst HSV U_(s)3-induced inhibition of apoptosis comprises the stepsof (a) providing a first cell comprising an HSV U_(s)3 gene under thecontrol of an inducible promoter; (b) inducing transcription from saidpromoter; (c) contacting said first cell with a test compound; (d)incubating said first cell under conditions expression of an HSV U_(s)3polypeptide; and (e) comparing the cell pathology of said first cellfollowing incubation with the cell pathology of a second cell not havingan HSV U_(s)3 gene following induction and the cell pathology of a thirdcell comprising an HSV U_(s)3 gene under the control of an induciblepromoter following induction but in the absence of said test compound.

Yet another screening method for compounds having inhibitory activityagainst HSV U_(s)3 polypeptide-induced inhibition of apoptosis comprisesthe steps of (a) providing a first cell; (b) infecting said first cellwith a herpes simplex virus; (c) contacting said first cell with a testcompound; (d) incubating said first cell under conditions permittingviral replication; and (e) comparing the cell pathology of said firstcell following incubation with the cell pathology of a second celltreated with said test compound alone and the cell pathology of a thirdcell following infection with said herpes simplex virus but in theabsence of said test compound.

In still yet a further embodiment, there is provided a method forexpressing a polypeptide in a cell comprising the steps of (a)contacting a cell with a herpes virus vector encoding said polypeptide;(b) contacting said cell with an agent that blocks the transactivatingfunction of U_(s)3 but does not block the apoptosis inhibiting functionof U_(s)3.

In yet still another embodiment, there is provided a screening methodfor compounds having inhibitory activity against HSV ICP4polypeptide-induced inhibition of apoptosis comprising the steps of (a)providing a first cell comprising an HSV α4 gene under the control of anHSV immediate early promoter; (b) infecting said first cell with aherpes simplex virus that lacks a functional α4 gene; (c) contactingsaid first cell with a test compound; (d) incubating said first cellunder conditions permitting viral replication; and (e) comparing thecell pathology of said first cell following incubation with the cellpathology of a second cell that lacks an HSV α4 gene following infectionwith said herpes simplex virus and the cell pathology of a third cellcomprising an HSV α4 gene under the control of an HSV immediate earlypromoter following infection with said herpes simplex virus but in theabsence of said test compound. Cell pathology comprises condensation ofchromatin, obliteration of nuclear membranes, vacuolization, cytoplasmicblebbing and DNA fragmentation.

The screening method may employ a cell line which contains an integratedcopy of a wild-type HSV α4 gene under the control of an α4 promoter anda herpes simplex virus that lacks a functional HSV α4 gene has adeletion in both copies of the virally-encoded α4 genes. For example,the herpes virus may carry a temperature sensitive mutation in bothcopies of the virally-encoded α4 gene; incubation is at 39.5° C.

Another screening method for compounds having inhibitory activityagainst HSV ICP4-induced inhibition of apoptosis comprises the steps of(a) providing a first cell comprising an HSV α4 gene under the controlof an inducible promoter; (b) inducing transcription from said promoter;(c) contacting said first cell with a test compound; (d) incubating saidfirst cell under conditions expression of an HSV ICP4 polypeptide; and(e) comparing the cell pathology of said first cell following incubationwith the cell pathology of a second cell not having an HSV α4 genefollowing induction and the cell pathology of a third cell comprising anHSV α4 gene under the control of an inducible promoter followinginduction but in the absence of said test compound.

Yet another screening method for compounds having inhibitory activityagainst HSV ICP4 polypeptide-induced inhibition of apoptosis comprisesthe steps of (a) providing a first cell; (b) infecting said first cellwith a herpes simplex virus; (c) contacting said first cell with a testcompound; (d) incubating said first cell under conditions permittingviral replication; and (e) comparing the cell pathology of said firstcell following incubation with the cell pathology of a second celltreated with said test compound alone and the cell pathology of a thirdcell following infection with said herpes simplex virus but in theabsence of said test compound.

In still yet a further embodiment, there is provided a method forexpressing a polypeptide in a cell comprising the steps of (a)contacting a cell with a herpes virus vector encoding said polypeptide;(b) contacting said cell with an agent that blocks the transactivatingfunction of ICP4 but does not block the apoptosis inhibiting function ofICP4.

The present invention also provides a method for the identification of acandidate substance that is a modulator of US3-kinase activitycomprising the steps of (a) providing an enzyme composition comprisingU_(s)3 protein kinase and a threonine/serine protein substrate; (b)contacting the composition with the candidate substance under conditionsthat allow phosphorylation; (c) determining the phosphorylation of thethreonine/serine substrate; and (d) comparing the phosphorylation of thethreonine/serine substrate with the phosphorylation of thethreonine/serine substrate in the absence of a candidate inhibitorsubstance; wherein an increase in phosphorylation is indicative of thecandidate substance being a stimulator of phosphorylation and a decreasein phosphorylation of the threonine/serine substrate is indicative ofthe candidate being an inhibitor of phosphorylation.

The present invention further provides an inhibitor of U_(s)3-kinaseactivity identified according to a method comprising the steps of (a)providing an enzyme composition comprising U_(s)3 protein kinase and athreonine/serine protein substrate; (b) contacting the composition withthe candidate substance under conditions that allow phosphorylation; (c)determining the phosphorylation of the threonine/serine substrate; and(d) comparing the phosphorylation of the threonine/serine substrate withthe phosphorylation of the threonine/serine substrate in the absence ofa candidate inhibitor substance; wherein a decrease in phosphorylationof the threonine/serine substrate is indicative of the candidate beingan inhibitor of phosphorylation.

In another embodiments there is provided a stimulator of U_(s)3-kinaseactivity identified according to a method comprising the steps of (a)providing an enzyme composition comprising U_(s)3 protein kinase and athreonine/serine protein substrate; (b) contacting said composition withsaid candidate substance under conditions that allow phosphorylation;(c) determining the phosphorylation of said threonine/serine substrate;and (d) comparing the phosphorylation of said threonine/serine substratewith the phosphorylation of said threonine/serine substrate in theabsence of a candidate inhibitor substance; wherein an increase inphosphorylation is indicative of said candidate substance being astimulator of phosphorylation.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Diagrammatic representation of the HSV-1 genome showing thelocation of the α4 and U_(L)29 genes encoding ICP4 and ICP8,respectively. The reiterated sequences (open rectangles) flanking theunique short (U_(S)) and unique long (U_(L)) sequences (thin lines) andthe location and direction of genes are as shown. Because the α4 genemaps within inverted repeats flanking the U_(L), it is present in twocopies per genome. The hatched lines within the rectangles indicates theposition of the sequences deleted from the d120 mutant (DeLuca, et al.,1985).

FIG. 2. Photograph of an agarose gel stained with ethidium bromide. Verocells were mock-infected (lane 1), infected with HSV-1(F) (lane 2),HSV-1 d120 mutant (lane 3), HSV-1 120FR mutant (lane 4), or HSV-1 120KRmutant (lane 5). 30 hr post infection, 2×10⁶ cells per sample werecollected, washed in PBS, lysed in a solution containing 10 mM Tris-HCI,pH 8.0, 10 mM EDTA, and 0.5% Triton X-100, and centrifuged at 12,000 rpmfor 25 min in an Eppendorf microcentrifuge to pellet chromosomal DNA.Supernatant fluids were digested with 0.1 mg RNase A per ml at 37° C.for 1 hr and then for 2 hr with 1 mg proteinase K per ml at 50° C. inthe presence of 1 % sodium dodecylsulphase (SDS), extracted with phenoland chloroform, and precipitated in cold ethanol and subjected toelectrophoresis on horizontal 1.5% agarose gels containing 5 μg ofethidium bromide per ml. DNA was visualized by UV lighttransillumination. Photographs were taken with the aid of acomputer-assisted image processor (Eagle Eye II®, Strategene).

FIG. 3. Photograph of an agarose gel stained with ethidium bromide. Verocells were mock-infected (lane 1), infected with HSV-1 120FR mutant(lane 2), wild-type HSV-1(F) (lane 3), HSV-1 R325 mutant (lane 4), orHSV-1 R7041 mutant (lane 5). Cells were harvested and processed asdescribed in the legend to FIG. 2.

FIG. 4. Photograph of an agarose gel stained with ethidium bromide. Verocells were infected with HSV-1(F) (lane 1), HSV-1 120FR mutant (lane 2),HSV-1 R7041 mutant (lane 3), HSV-1 R7306 mutant (lane 4), ordouble-infected with HSV-1 120FR and R7041 mutants (lane 5), HSV-1 d120and 120FR mutants (lane 6), or HSV-1 120FR and R7306 mutants (lane 7).Cells were harvested and processed as described in the legend to FIG. 2.

FIG. 5. BHK-C13 cells were infected at a multiplicity of 10 pfu/cellwith the indicated viruses. At 13 hr post infection cells were incubatedin phosphate-free medium for 1 hr and then labeled with 32Pi for 4 hr.Cell lysates were electrophoretically separated in SDS polyacrylarnidegels and electrically transferred onto nitrocellulose filters. Filterswere exposed to film or probed with a UL34 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The identification of novel proteins having apoptotic activities anduses will provide important new tools for those working with thechallenge of treatment and prevention of HSV infection. The presentinvention provides methods for the use of Us3 gene and its gene productas inhibitors of apoptosis.

The herpes simplex virus 1 (HSV-1) mutant, which lacks the majorregulatory gene designated α4, induced apoptosis, whereas in cellsinfected with wild-type virus, apoptosis did not ensue (Leopardi andRoizman, 1996). The inventors also reported that wild type virus blockedapoptosis induced by thermal shock. Recent studies by Koyama and Miwa(1997) and also in the inventors' laboratory demonstrated that the virusalso blocks the induction of apoptosis by osmotic shock. These studiessuggested that a functional α4 gene was necessary to block apoptosis butdid not address the question of sufficiency. This led the inventors todesign several studies that included the rescue of the deleted α4 genesin the mutant virus.

The inventors discovered that the mutant lacking the α4 gene,HSV-1(KOS)d120, had a secondary mutation in the gene U_(s)3 specifying aprotein kinase. Thus, the inventors have discovered that a functionalU_(s)3 is required in order to block apoptosis. This important findingmay now be exploited in the inhibition of apoptosis, the induction ofapoptosis, as described herein below. Further, the present inventionallows for the provision of methods for the use of agents that inhibitU_(s)3 and the U_(s)3 gene in order to induce apoptosis in HSV infectedcells and for the identification of agents that inhibit theapoptosis-inhibiting function of ICP4. These and other related aspectsof the present invention are described in further detail herein below.

I. Herpes Simplex Virus

Herpes simplex viruses, designated with subtypes 1 and 2, are envelopedviruses that are among the most common infectious agents encountered byhumans, infecting millions of human subjects worldwide. These virusescause a broad spectrum of disease which ranges from relativelyinsignificant to severe and life-threatening. The clinical outcome ofherpes infections is dependent upon early diagnosis and promptinitiation of antiviral therapy. Despite some successful efforts intreating HSV infectious, dermal and epidermal lesion often recur, andHSV infections of neonates and infections of the brain are associatedwith high morbidity and mortality.

The large, complex, double-stranded DNA genome encodes dozens ofdifferent gene products, some of which derive from spliced transcripts.In addition to virion and envelope structural components, the virusencodes numerous other proteins including a protease, a ribonucleotidesreductase, a DNA polymerase, a ssDNA binding protein, ahelicase/primase, a DNA dependent ATPase, a dUTPase and others.

HSV genes form several groups whose expression is coordinately regulatedand sequentially ordered in a cascade fashion (Honess and Roizman, 1974;Honess and Roizman 1975; Roizman and Sears, 1996). The expression of αgenes, the first set of genes to be expressed after infection, isenhanced by the virion protein number 16, or α-transinducing factor(Post et al., 1981; Batterson and Roizman, 1983; Campbell, et al.,1984). The expression of β genes requires functional α gene products,most notably ICP4, which is encoded by the α4 gene (DeLuca et al.,1985). γ genes, a heterogeneous 25 group of genes encoding largelyvirion structural proteins, require the onset of viral DNA synthesis foroptimal expression (Holland et al., 1980).

In line with the complexity of the genome, the life cycle of HSV isquite involved. In addition to the lytic cycle, which results insynthesis of virus particles and, eventually, cell death, the virus hasthe capability to enter a latent state in which the genome is maintainedin neural ganglia until some as of yet undefined signal triggers arecurrence of the lytic cycle.

II. The ICP4 Polypeptide

As stated above, the expression of P genes is regulated in a majorfashion by ICP4 (DeLuca et al., 1985), and therefore this gene has adistinct effect on viral DNA synthesis. γ genes, a heterogeneous groupof genes encoding largely virion structural proteins, require the onsetof viral DNA synthesis for optimal expression (Honess and Roizman, 1975;Holland et al., 1980). ICP4 plays a key role in this process—cellsinfected with viruses carrying temperature sensitive mutations in the a4gene and maintained at nonpermissive temperatures express largely aproteins (Dixon and Shaffer, 1980). Furthermore, studies involvingshift-up of infected cells from permissive to nonpermissive temperatureshave confirmed a key role for ICP4 throughout the viral reproductivecycle.

ICP4 acts both as a transactivator and as a repressor (Roizman andSears, 1996). The response elements for the repressor functions of ICP4are high affinity binding sites located in the proximity oftranscription initiation sites of the genes repressed by the protein(Kristie and Roizman, 1986a, Kristie and Roizman, 1986b, Faber andWilcox, 1986, Muller, 1987, Michael and Roizman, 1989, Michael andRoizman, 1993). The strength of repression is dependent on both thedistance and stereoaxial alignment with the TATA box (Leopardi et al.,1995, Kuddus et al., 1995). Low affinity sites for binding of ICP4 havealso been documented, but their function is less well understood(Kristie and Roizman, 1986a, Kristie and Roizman, 1986b, Michael et al.,1988). The response elements thought to act in the transactivation ofviral genes by ICP4 are not known, but mutations in ICP4 may affectrepression and activation independently of each other (Shepard andDeLuca, 1991).

Thus, the repressor function of ICP4 is associated with the presence ofhigh affinity sites located at or near the transcription initiationsites of several genes. The transactivating function has not beenassociated to specific binding to DNA although it has been shown thatlate viral gene expression requires the association of ICP4 with nascentviral DNA and several host (e.g., RNA polymerase II, Epstein Barr virussmall nuclear RNA associated host protein) proteins and ICP22, a viraltranscriptional factor (Leopardi et al., 1997). Extensive studies withdeletion mutants exemplified by HSV-1(KOS)d120 led DeLuca et al. (1985)to map distinct domains within the ICP4 proteins. Studies in theinventors' laboratory have shown that ICP4 is extensively modified posttranslationally by phosphorylation, ADP(ribosyl)ation, andnucleotidylylation (Roizman and Sears, 1996). The expectation is thatthese modifications are not random effects by existing enzymes but thateach post translational modification enhances or inactivates a specificfunction of the protein.

III. The U_(s)3 Polypeptide

HSV encodes two protein kinases expressed by the genes U_(s)3 andU_(L)13, respectively (reviewed in Roizman and Sears, 1996). WhereasU_(L)13 is packaged in the virion, U_(s)3 is not. Not all substrates ofthe U_(s)3 are known (Purves et al., 1986; 1987; Leader et al., 1991).The major substrate of U_(s)3 protein kinase is an intrinsic membraneprotein exposed on the surface of infected cells and encoded by theU_(L)34 gene (Purves et al., 1991; 1992). The U_(L)34 is phosphorylatedby more than one kinase (Purves 1991, 1992). In the absence of U_(s)3protein kinase, the U_(L)34 protein has an apparent M_(r) of 33,000 andassociates with several cellular phosphoproteins whereas in wild typeinfected cells U_(L)34 has an apparent M_(r) of 30,000 and does notexhibit an association with the host proteins (Purves et al., 1992).

The U_(s)3 protein kinase phosphorylates theronine/serine in theconsensus sequence RRR-R/X-S/T-RIY (SEQ D NO:6) (Purves et al., 1986;Leader et al., 1991). In the case of U_(L)34, this was verified bymutagenesis of the threonine codon in the sequence encoding RRRRTRRSRE(SEQ ID NO:5) (Purves et al., 1991; 1992). The apparent M_(r) of themutant U_(L)34 protein was 33,000, that is, corresponding to theapparent M_(r) of the U_(L)34 protein in cells infected with a viruslacking the U_(s)3 gene.

The inventors predict that U_(s)3 blocks apoptosis by phosphorylatingone or more proteins. As stated above, for ICP4 to be functional itneeds to be phosphorylated, this leads the inventors to believe that oneof the substrates of U_(s)3 protein kinase is ICP4 itself, among other,as yet unidentified, phosphoproteins.

The involvement of U_(s)3 protein kinase in blocking of apoptosisinduced by infection, thermal or osmotic shocks suggests that HSVdiffers from other viruses in the mechanism by which it blocks theapoptosis as a host response to infection. Thus, U_(s)3 may be used toin control apoptosis independent of viral infection.

Accordingly, the present invention describes a novel functional aspectof the protein kinase U_(s)3 that was previously undisclosed is itsability to inhibit apoptosis. Further, the invention describes a novelfunctional aspect of ICP4 as an inhibitor of apoptosis. Apoptosis, orprogrammed cell death, is characterized by certain cellular events,including nuclear condensation, DNA fragmentation, cytoplasmic membraneblebbing and, ultimately, irreversible cell death. Apoptosis is anenergy-dependent event. For the purposes of this application, apoptosiswill be defined as inducing one or more of these events. Thus, use ofthe term “U_(s)3” in this application encompasses polypeptides havingthe anti-apoptosis function of U_(s)3. These need not be wild-typeU_(s)3. Likewise, the use of the term “ICP4” in this applicationencompasses polypeptides having the anti-apoptosis function of ICP4 andit need not be wild-type ICP4.

This functional attribute is manifested, for example, in U_(x)3's orICP4's ability, alone or in combination with one another, to protectcells from apoptosis triggered by modification of cellular physiology byother viral genes. This observation permits utilization of U_(s)3 orICP4 alone or in combination with each other, in a number of ways thatcould not have been predicted from the prior art. For example, accordingto the present invention, the production of HSV vectors or recombinantproteins from HSV vectors can be enhanced by increasing the apoptosisinhibiting function of U_(s)3 or ICP4 or both. When cells are infectedwith HSV, premature cell death can limit the titer of virus produced orthe amount of recombinant protein synthesized. Similarly, U_(s)3 andICP4 may prolong the life of the cells expressing human or animal genesintroduced into cells by viral vectors in order to correct geneticdefect. If the cell can be sustained longer, the titer of the virusstocks and the amount of protein should increase.

U_(s)3 and ICP4 may be obtained according to various standardmethodologies that are known to those of skill in the art. For example,antibodies specific for U_(s)3 or ICP4 may be used in immunoaffinityprotocols to isolate the respective polypeptide from infected cells, inparticular, from infected cell lysates. Antibodies are advantageouslybound to supports, such as columns or beads, and the immobilizedantibodies can be used to pull the U_(s)3 or IPC4 target out of the celllysate.

Alternatively, expression vectors, rather than viral infections, may beused to generate the polypeptide of interest. A wide variety ofexpression vectors may be used, including viral vectors. The structureand use of these vectors is discussed further, below. Such vectors maysignificantly increase the amount of U_(s)3 and/or ICP4 protein in thecells, and may permit less selective purification methods such as sizefractionation (chromatography, centrifugation), ion exchange or affinitychromatograph, and even gel purification. Alternatively, the expressionvector may be provided directly to target cells, again as discussedfurther, below.

U_(s)3, according to the present invention, may advantageously becleaved into fragments for use in further structural or functionalanalysis, or in the generation of reagents such as U_(s)3-relatedpolypeptides and U_(s)3-specific antibodies. This can be accomplished bytreating purified or unpurified U_(s) ³ with a peptidase such asendoproteinase glu-C (BOEHRINGER®, Indianapolis, Ind.). Treatment withCNBr is another method by which U_(s)3 fragments may be produced fromnatural U_(s)3. Recombinant techniques also can be used to producespecific fragments of U_(s)3. It may be that the phosphorylating andapoptosis-inhibiting functions of U_(s)3 reside in distinct domains ofthe protein. If such is the case, the ability to make domain-specificreagents now has significance. For example, the ability to provide anapoptosis-inhibiting U_(s)3 fragment that does not phosphorylate viralgenes may prove to be effective in extending the life of neuronsexpressing compensatory or therapeutic genes from a viral vector.

Likewise ICP4, may advantageously be cleaved into fragments for use infurther structural or functional analysis, or in the generation ofreagents such as ICP4-related polypeptides and ICP4-specific antibodies.Because the transactivating and apoptosis-inhibiting functions of ICP4appear to reside in distinct domains, the ability to makedomain-specific reagents now has significance. For example, the abilityto provide an apoptosis-inhibiting ICP4 fragment that does nottransactivate viral genes may prove to be effective in extending thelife of neurons expressing compensatory or therapeutic genes from aviral vector.

It is expected that changes may be made in the sequence of U_(s)3 orICP4 while retaining a molecule having the structure and function of thenatural U_(s)3 or ICP4 respectively. For example, certain amino acidsmay be substituted for other amino acids in a protein structure withoutappreciable loss of interactive capacity with structures such as, forexample, substrate-binding regions. These changes are termed“conservative” in the sense that they preserve the structural and,presumably, required functional qualities of the starting molecule. Theimportance of U_(s)3 variants is highlighted by the observation,discussed in the examples, that mutants lacking the α4 gene also have anadditional mutation in the US3 gene making it non-functional inapoptosis. The mutation in U_(s)3 was silent and would not have affectedthe studies carried out by DeLuca et al. (1985) since viral geneexpression, including that of U_(s)3, requires a functional a4 gene andapoptosis is a very late event. Even if a rescue had been done, it wouldnot have detected the mutation in U_(s)3 unless the infected cell wereprobed for the U_(s)3 function.

The importance of ICP4 variants is highlighted by the observation thattemperature sensitive (ts) mutants of ICP4 exist that are impaired intheir ability to transactivate viral genes at elevated temperatures(above about 39° C.), but retain the apoptosis inhibiting functionassociated with this polypeptide. Further exploration of this dichotomyshould reveal significant information on the regions in which thesefunctions lie. It has been shown that the transactivation domain of ICP4lies between about residues 100 and 200, the DNA-binding domains liesbetween about residues 300 and 500, the nuclear localization domain liesbetween about residues 700 and 750 and the transactivation domain liesbetween about residues 750 and 1298.

Conservative amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. An analysis of the size, shape and type of the amino acidside-chain substituents reveals that arginine, lysine and histidine areall positively charged residues; that alanine, glycine and serine areall a similar size; and that phenylalanine, tryptophan and tyrosine allhave a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and histidine; alanine, glycine andserine; and phenylalanine, tryptophan and tyrosine; are defined hereinas equivalent.

In making such changes, the hydropathic index of amino acids also may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982). It is known that certain amino acidsmay be substituted for other amino acids having a similar hydropathicindex or score and still retain a similar biological activity. In makingchanges based upon the hydropathic index, the substitution of aminoacids whose hydropathic indices are within ±2 is preferred, those whichare within ±1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the polypeptide created is intended for use inimmunological embodiments, as in the present case. U.S. Pat. No.4,554,101, incorporated herein by reference, states that the greatestlocal average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e., with a biological property of theprotein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0 ±1); glutamate (+3.0 ±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

Numerous scientific publications have been devoted to the prediction ofsecondary structure, and to the identification of epitopes, fromanalyses of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b;1979). Any of these may be used, if desired, to supplement the teachingsof Hopp in U.S. Pat. No. 4,554,101. Moreover, computer programs arecurrently available to assist with predicting antigenic portions andepitopic core regions of proteins. Examples include those programs basedupon the Jameson-Wolf analysis (Jameson & Wolf, 1988; Wolf et al.,1988), the program PEPPLOT® (Brutlag et al., 1990; Weinberger et al.,1985), and other new programs for protein tertiary structure prediction(Fetrow & Bryant, 1993).

Two designations for amino acids are used interchangeably throughoutthis application, as is common practice in the art. Alanine=Ala (A);Arginine=Arg (R); Aspartate=Asp (D); Asparagine=Asn (N); Cysteine=Cys(C); Glutamate=Glu (E); Glutamine=Gln (Q); Glycine=Gly (G);Histidine=His (H); Isoleucine=Ile (I); Leucine =Leu (L); Lysine=Lys (K);Methionine=Met (M); Phenylalanine=Phe (F); Proline=Pro (P); Serine=Ser(S); Threonine=Thr (T); Tryptophan=Trp (W); Tyrosine=Tyr (Y); Valine=Val(V).

In addition to the peptidyl compounds described herein, the inventorsalso contemplate that other sterically similar compounds may beformulated to mimic the key portions of the peptide structure, calledpeptidomimetics. Mimetics are peptide-containing molecules which mimicelements of protein secondary structure. See, for example, Johnson etal. (1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of receptor and ligand.

Successful applications of the peptide mimetic concept have thus farfocused on mimetics of β-turns within proteins. Likely β-turn structureswithin U_(s)3 and ICP4 can be predicted by computer-based algorithms asdiscussed above. Once the component amino acids of the turn aredetermined, mimetics can be constructed to achieve a similar spatialorientation of the essential elements of the amino acid side chains, asdiscussed in Johnson et al., supra.

IV. Nucleic Acids Encoding U_(s)3 and ICP4

Also contemplated by the present invention are nucleic acids encodingU_(s)3 and ICP4. The gene for US3 is given in SEQ ID NO:3. The gene forICP4 is known as α4 (SEQ ID NO: 1). The amino acid sequences for U_(s)3and ICP4 encoded by these genes are given in SEQ ID NO:4 and SEQ IDNO:2, respectively. The full length genomic sequence of HSV is known andcan be found in Genbank (Accession No. x14112); ICP4 is encoded byidentical diploid genes inverted relative to each other, their codingsequences are located from nucleotide 131, 128 to 127, 232 and 147,104to 151, 000. The US3 protein coding sequence is from nucleotide 135, 222to 136, 667. Because of the degeneracy of the genetic code, many othernucleic acids also may encode a given U_(s)3 or a given ICP4. Forexample, four different three-base codons encode the amino acidsalanine, glycine, proline, threonine and valine, while six differentcodons encode arginine, leucine and serine. Only methionine andtryptophan are encoded by a single codon. A table of amino acids and thecorresponding codons is presented herein for use in such embodiments.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In order to generate any nucleic acid encoding U_(s)3 or ICP4, one needonly refer to the preceding codon table. Substitution of the naturalcodon with any codon encoding the same amino acid will result in adistinct nucleic acid that encodes U_(s)3 or ICP4 or a variant thereof.As a practical matter, this can be accomplished by site-directedmutagenesis of an existing U_(s)3 or α4 gene or de novo chemicalsynthesis of one or more nucleic acids.

The preceding observations regarding codon selection, site-directedmutagenesis and chemical synthesis apply with equal force to thediscussion of substitutional mutants in the section of peptides.Normally, substitutional mutants are generated by site-directed changesin the nucleic acid designed to alter one or more codons of the codingsequence.

In order to express an U_(s)3 polypeptide, or an antisense U_(s)3transcript, it is necessary to provide an U_(s)3 gene in an expressionvehicle. Similarly to express an ICP4 polypeptide, or an antisense α4transcript, it is necessary to provide an α4 gene in an expressionvehicle. The appropriate nucleic acid can be inserted into an expressionvector by standard subcloning techniques. For example, an E. coli orbaculovirus expression vector is used to produce recombinant polypeptidein vitro. The manipulation of these vectors is well known in the art. Inone embodiment, the protein is expressed as a fusion protein with β-gal,allowing rapid affinity purification of the protein. Examples of suchfusion protein expression systems are the glutathione S-transferasesystem (PHARMACIA, Piscataway, N.J.), the maltose binding protein system(NEB, Beverley, Mass.), the FLAG® system (IBI®, New Haven, Conn.), andthe 6xHis® system (QIAGEN, Chatsworth, Calif.).

Some of these fusion systems produce recombinant protein bearing only asmall number of additional amino acids, which are unlikely to affect thefunctional capacity of the recombinant protein. For example, both theFLAG® system and the 6xHIS® system add only short sequences, both ofwhich are known to be poorly antigenic and which do not adversely affectfolding of the protein to its native conformation. Other fusion systemsproduce proteins where it is desirable to excise the fusion partner fromthe desired protein. In another embodiment, the fusion partner is linkedto the recombinant protein by a peptide sequence containing a specificrecognition sequence for a protease. Examples of suitable sequences arethose recognized by the Tobacco Etch Virus protease (LIFE TECHNOLOGIES®,Gaithersburg, Md.) or Factor Xa (NEW ENGLAND BIOLABS, Beverley, Mass.).

Recombinant bacterial cells, for example E. coli, are grown in any of anumber of suitable media, for example LB, and the expression of therecombinant polypeptide induced by adding IPTG to the media or switchingincubation to a higher temperature. After culturing the bacteria for afurther period of between 2 and 24 hours, the cells are collected bycentrifugation and washed to remove residual media. The bacterial cellsare then lysed, for example, by disruption in a cell homogenizer andcentrifuged to separate the dense inclusion bodies and cell membranesfrom the soluble cell components. This centrifugation can be performedunder conditions whereby the dense inclusion bodies are selectivelyenriched by incorporation of sugars such as sucrose into the buffer andcentrifugation at a selective speed.

If the recombinant protein is expressed in the inclusion bodies, as isthe case in many instances, these can be washed in any of severalsolutions to remove some of the contaminating host proteins, thensolubilized in solutions containing high concentrations of urea (e.g.8M) or chaotropic agents such as guanidine hydrochloride in the presenceof reducing agents such as B-mercaptoethanol or DTT (dithiothreitol).

Under some circumstances, it may be advantageous to incubate thepolypeptide for several hours under conditions suitable for the proteinto undergo a refolding process into a conformation which more closelyresembles that of the native protein. Such conditions generally includelow protein concentrations less than 500 μg/ml, low levels of reducingagent, concentrations of urea less than 2 M and often the presence ofreagents such as a mixture of reduced and oxidized glutathione whichfacilitate the interchange of disulphide bonds within the proteinmolecule.

The refolding process can be monitored, for example, by SDS-PAGE or withantibodies which are specific for the native molecule (which can beobtained from animals vaccinated with the native molecule isolated fromparasites). Following refolding, the protein can then be purifiedfurther and separated from the refolding mixture by chromatography onany of several supports including ion exchange resins, gel permeationresins or on a variety of affinity columns.

In yet another embodiment, the expression system used is one driven bythe baculovirus polyhedron promoter. The gene encoding the protein canbe manipulated by standard techniques in order to facilitate cloninginto the baculovirus vector. A preferred baculovirus vector is thepBlueBac vector (INVITROGEN®, Sorrento, Calif.). The vector carrying thegene of interest is transfected into Spodoptera frugiperda (Sf9) cellsby standard protocols, and the cells are cultured and processed toproduce the recombinant protein.

There also are a variety of eukaryotic vectors that provide a suitablevehicle in which recombinant polypeptide can be produced. HSV itself hasbeen used in tissue culture to express a large number of exogenous genesas well as for high level expression of its endogenous genes. Forexample, the chicken ovalbumin gene has been expressed from HSV using ana promoter. Herz and Roizman (1983). The lacZ gene also has beenexpressed under α variety of HSV promoters.

In an alternative embodiment, the nucleic acids employed actually mayencode antisense constructs that hybridize, under intracellularconditions, to an US3 nucleic acid or an α4 nucleic acid. The term“antisense construct” is intended to refer to nucleic acids, preferablyoligonucleotides, that are complementary to the base sequences of atarget DNA or RNA. Antisense oligonucleotides, when introduced into atarget cell, specifically bind to their target nucleic acid andinterfere with transcription, RNA processing, transport, translationand/or stability.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNA's,may be employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject. Nucleic acid sequences which comprise“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA. Inclusion of less common basessuch as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine andothers in hybridizing sequences does not interfere with pairing.

As used herein, the term “complementary” means nucleic acid sequencesthat are substantially complementary over their entire length and havevery few base mismatches. For example, nucleic acid sequences of fifteenbases in length may be termed complementary when they have acomplementary nucleotide at thirteen or fourteen positions with only asingle mismatch. Naturally, nucleic acid sequences which are “completelycomplementary” will be nucleic acid sequences which are entirelycomplementary throughout their entire length and have no basemismatches.

Other sequences with lower degrees of homology also are contemplated.For example, an antisense construct which has limited regions of highhomology, but also contains a non-homologous region (e.g., a ribozyme)could be designed. These molecules, though having less than 50%homology, would bind to target sequences under appropriate conditions.

While all or part of the gene sequence of interest may be employed inthe context of antisense construction, short oligonucleotides are easierto make and increase in vivo accessibility. However, both bindingaffinity and sequence specificity of an antisense oligonucleotide to itscomplementary target increases with increasing length. It iscontemplated that antisense oligonucleotides of 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100or more base pairs will be used. One can readily determine whether agiven antisense nucleic acid is effective at targeting of thecorresponding host cell gene simply by testing the constructs in vitroto determine whether the endogenous gene's function is affected orwhether the expression of related genes having complementary sequencesis affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., 1990).

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. Thus, in certainembodiments, expression includes both transcription of a gene andtranslation of a RNA into a gene product. In other embodiments,expression only includes transcription of the nucleic acid, for example,to generate antisense constructs.

In preferred embodiments, the nucleic acid is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of a gene.The phrase “under transcriptional control” means that the promoter is inthe correct location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell. Generally speaking, such a promotermight include either a human or viral promoter. Preferred promotersinclude those derived from HSV, including the U_(s)3, or the α4promoter. Another preferred embodiment is the tetracycline controlledpromoter.

In various other embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter and the Rous sarcoma viruslong terminal repeat can be used to obtain high-level expression oftransgenes. The use of other viral or mammalian cellular or bacterialphage promoters which are well-known in the art to achieve expression ofa transgene is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose. Tables 2 and 3 listseveral elements/promoters which may be employed, in the context of thepresent invention, to regulate the expression of a transgene. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of transgene expression but, merely, to be exemplarythereof.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression of atransgene. Use of a T3, T7 or SP6 cytoplasmic expression system isanother possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

TABLE 2 PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light ChainT-Cell Receptor HLA DQ α and DQ β β-Interferon Interleukin-2Interleukin-2 Receptor MHC Class II 5 MHC Class II HLA-DRα β-ActinMuscle Creatine Kinase Prealbumin (Transthyretin) Elastase IMetallothionein Collagenase Albumin Gene α-Fetoprotein τ-Globin β-Globinc-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)α_(1-Antitrypsin) H2B (TH2B) Histone Mouse or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone HumanSerum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth FactorDuchenne Muscular Dystrophy SV40 Polyoma Retroviruses Papilloma VirusHepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus GibbonApe Leukemia Virus

TABLE 3 Element Inducer MT II Phorbol Ester (TPA) Heavy metals MMTV(mouse mammary tumor Glucocorticoids virus) β-Interferon poly(rI)Xpoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 PhorbolEster (TPA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kBInterferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPATumor Necrosis Factor FMA Thyroid Stimulating Hormone α Thyroid HormoneGene

Use of the baculovirus system will involve high level expression fromthe powerful polyhedron promoter.

One will typically include a polyadenylation signal to effect properpolyadenylation of the transcript. The nature of the polyadenylationsignal is not believed to be crucial to the successful practice of theinvention, and any such sequence may be employed. Preferred embodimentsinclude the SV40 polyadenylation signal and the bovine growth hormonepolyadenylation signal, convenient and known to function well in varioustarget cells. Also contemplated as an element of the expression cassetteis a terminator. These elements can serve to enhance message levels andto minimize read through from the cassette into other sequences.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancer elements(Bittner et al., 1987).

In various embodiments of the invention, the expression construct maycomprise a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and to integrate into host cell genome and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign genes into mammalian cells (Ridgeway, 1988;Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).The first viruses used as vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986) and adeno-associated viruses.Retroviruses also are attractive gene transfer vehicles (Nicolas andRubenstein, 1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988) andadeno-associated virus (Ridgeway, 1988). Such vectors may be used to (i)transform cell lines in vitro for the purpose of expressing proteins ofinterest or (ii) to transform cells in vitro or in vivo to providetherapeutic polypeptides in a gene therapy scenario.

In a preferred embodiment, the vector is HSV. Because HSV isneurotropic, it has generated considerable interest in treating nervoussystem disorders. Moreover, the ability of HSV to establish latentinfections in non-dividing neuronal cells without integrating into thehost cell chromosome or otherwise altering the host cell's metabolism,along with the existence of a promoter that is active during latency.And though much attention has focused on the neurotropic applications ofHSV, this vector also can be exploited for other tissues.

Another factor that makes HSV an attractive vector is the size andorganization of the genome. Because HSV is large, incorporation ofmultiple genes or expression cassettes is less problematic than in othersmaller viral systems. In addition, the availability of different viralcontrol sequences with varying performance (temporal, strength, etc.)makes it possible to control expression to a greater extent than inother systems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations.

HSV also is relatively easy to manipulate and can be grown to hightiters. Thus, delivery is less of a problem, both in terms of volumesneeded to attain sufficient MOI and in a lessened need for repeatdosings. For a review of HSV as a gene therapy vector, see Glorioso etal., 1995.

V. Methods for Screening

A. Inhibitors of U_(s)3 and ICP4 Anti-Apoptotic Activity

In one embodiment of the present invention, there are provided methodsof screening compounds for activity against U_(s)3's anti-apoptoticactivity. These screening methods will determine the cell pathology oftarget cells that express U_(s)3, both in the presence and absence ofthe test compound. In another embodiment, the present invention,provides methods of screening compounds for activity against ICP4'santi-apoptotic activity. At least three different assays may beemployed, as discussed below.

First, one may look at DNA fragmentation using a separative method,e.g., chromatography or electrophoresis, to size fractionate the sample.As described in greater detail in the examples, an exemplary assayinvolves the isolation of DNA from cells, followed by agarose gelelectrophoresis and staining with ethidium bromide. DNA fragmentation,characteristic of apoptosis, will be visualized as “ladders” containinga wide range of fragment sizes.

Second, one may employ terminal deoxynucleotidyl transferase mediateddUTP-biotin nick end labeling (TUNEL) assays to measures the integrityof DNA (Gorczyca, 1993). This assay measures the fragmentation of DNA bymonitoring the incorporation of labeled UTP into broken DNA strands bythe enzyme terminal transferase. The incorporation can be monitored byelectroscopy or by cell sorting methodologies (e.g., FACS)

Third, one may examine cells using standard light or electron microscopyto assess the presence or absence of the cytopathologies characteristicof apoptosis. Those of skill in the art, applying standard methods ofmicroscopy, will be able to assess cytopathology.

In each of these assays, a cell will be employed as the target forinduction and inhibition of apoptosis. In one embodiment, the cell willbe infected with HSV that expresses its own U_(s)3 protein. In a secondembodiment, the cell will carry the U_(s)3 gene linked to a viralpromoter. Infection with the appropriate virus will result instimulation of the U_(s)3 gene and expression of U_(s)3. In these firsttwo embodiments, the infection should induce apoptosis in the cell, andthe expression of US3 should limit this effect. In a third embodiment,the cell will contain, as part of its own genetic material, an inducibleversion of the U_(s)3 gene (i.e., U_(s)3 linked to an induciblepromoter). In this situation, it will be necessary to induce apoptosisvia some other mechanism, such as hypothermia, osmotic shock or ICP4expression, and express U_(s)3 by inducing the promoter. The presentinvention may further employ ICP4 alone or in combination with U_(s)3 inany of the embodiments described above.

The cell is contacted with a candidate inhibitor substance in order toassess its effect on U_(s)3 activity. The substance may be contactedwith the cell prior to, at the same time, or after the provision ofU_(s)3. In some cases, the candidate inhibitor substance may becontacted with the cell directly. In other situations, depending on thenature and putative mechanism of action, the candidate inhibitorsubstance may be reformulated to provide improved uptake. For example,where antisense oligonucleotides are provided, these may advantageouslybe formulated in liposomes or as virally-encapsulated expressionvehicles. Where polypeptides are to be tested, it may be advantageous toprovide expression vectors encoding these molecules rather than thepolypeptides themselves. Essentially, the most reasonable mechanism fordelivering an effective amount of the candidate inhibitor substance tothe proper intracellular site will be chosen. “Effective amount,” forthe purposes of the screening assay, is intended to mean an amount thatwill cause a detectable difference, and preferably a significantdifference, in the cytopathology of the cell as compared to a similartreatment of the cell without the candidate inhibitor substance. Asimilar protocol may be used to test the effect of a candidate substanceon ICP4 activity.

Once the candidate inhibitor substance has been provided to a cell thatexpresses the relevant peptide(s) (U_(s)3, ICP4 or both), the evaluationof cytopathology may be undertaken. Depending on the type of assay usedto examine cytopathology, it is possible to automate this process andtest hundreds of candidates at the same time. For example, 96-well traysmay be employed in which several wells are reserved for controls whilethe remainder comprise test substances, usually with each substancebeing tested at several different amounts.

B. Screening for Modulators of US3function

In certain embodiments, the present invention concerns a method foridentifying modulators of U_(s)3 function. Such modulators may beinhibitors or stimulators of such a function. It is contemplated thatthis screening technique will prove useful in the general identificationof any compound that will serve the purpose of inhibiting or stimulatingU_(s)3-mediated kinase activity.

The active compounds may include fragments or parts ofnaturally-occurring compounds or may be only found as activecombinations of known compounds which are otherwise inactive. However,prior to testing of such compounds in humans or animal models, it willpossibly be necessary to test a variety of candidates to determine whichhave potential.

Accordingly, in screening assays to identify pharmaceutical agents whichmodulate U_(s)3 kinase activity, it is proposed that compounds isolatedfrom natural sources, such as animals, bacteria, fungi, plant sources,including leaves and bark, and marine samples may be assayed ascandidates for the presence of potentially useful pharmaceutical agents.It will be understood that the pharmaceutical agents to be screenedcould also be derived or synthesized from chemical compositions orman-made compounds.

In these embodiments, the present invention is directed to a method fordetermining the ability of a candidate substance to inhibit or stimulatea protein kinase assay, the method including generally the steps of:

(a) obtaining an enzyme composition comprising a protein kinase,preferably U_(s)3 protein kinase that is capable of phosphorylatingthreonine/serine residue;

(b) admixing a candidate substance with the enzyme composition; and

(c) determining the ability of the candidate substance to inhibit orstimulate threonine/serine phosphorylation

To identify a candidate substance as being capable of modulating proteinphosphorylation, one would first obtain an enzyme composition that iscapable of phosphorylating threonine/serine residues on a protein ofinterest. Naturally, one would measure or determine the phosphorylationactivity of the threonine/serine kinase composition in the absence ofthe added candidate substance. One would then add the candidatesubstance to the threonine/serine kinase composition and re-determinethe ability of the threonine/serine kinase composition to phosphorylatethreonine/serine residues on a test protein in the presence of thecandidate substance. A candidate substance which reduces thephosphorylation activity of the threonine/serine kinase compositionrelative to the activity in its absence is indicative of a candidatesubstance with inhibitor capability. Likewise, a candidate substancewhich increases the phosphorylation activity of the threonine/serinekinase composition relative to the activity in its absence is indicativeof a candidate substance with stimulatory capability

The candidate screening assay is quite simple to set up and perform,after obtaining a relatively purified preparation of the enzyme, eitherfrom native or recombinant sources, one will admix a candidate substancewith the enzyme preparation, under conditions which would allow theenzyme to perform its threonine/serine phosphorylation function but forinclusion of a candidate substance. In this fashion, one can measure theability of the candidate substance to reduce or increase thethreonine/serine phosphorylation activity relatively in the presence ofthe candidate substance.

“Effective amounts” in certain circumstances are those amounts effectiveto reproducibly reduce or increase threonine/serine kinase activity, orto inhibit or induce the apoptosis of cells, in comparison to theirnormal levels. Compounds that achieve significant appropriate changes inactivity will be used. If desired, a battery of compounds may bescreened in vitro to identify agents for use in the present invention.

Significant decrease in threonine/serine phosphorylation, e.g., asmeasured using immunoblotting techniques with anti-phosphorylationantibodies, are represented by a reduction in protein phosphorylationlevels of at least about 30%-40%, and most preferably, by decreases ofat least about 50%, with higher values of course being possible.Threonine/serine kinase assays that measure threonine/serinephosphorylation are well known in the art and may be conducted in vitroor in vivo. Likewise increases in threonine/serine phosphorylation arerepresented by an increase in protein phosphorylation levels of at leastabout 30%-40%, and most preferably, by increases of at least about 50%,with higher values of course being possible.

Assays for apoptosis will measure certain cellular events, includingnuclear condensation, DNA fragmentation, cytoplasmic membrane blebbingand, ultimately, irreversible cell death. The methodology for suchmeasurements is well known to those of skill in the art. A significantalteration in apoptosis is represented by an increase or decrease of atleast about 30%-40% as compared to normal, and most preferably, of atleast about 50%, with more significant increases or decreases also beingpossible. Therefore, if a candidate substance exhibited inhibition orinduction of apoptosis in this type of study, it would likely be asuitable compound for use in the present invention.

Quantitative in vitro testing is not a requirement of the invention asit is generally envisioned that the candidate substance will often beselected on the basis of their known properties or by structural and/orfunctional comparison to those agents already known to have an effect onthreonine/serine kinases.

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

VI. Methods for the Inhibition of Apoptosis

In one embodiment of the present invention, there are provided methodsfor the inhibition of apoptosis in a cell. This is particularly usefulwhere one seeks to immortalize a cell or, at a minimum, increase thelongevity of a cell. This permits one to maintain that cell in culturefor extended periods of time, perhaps indefinitely. Immortalized cellsare useful primarily as factories for production of viral vectors orproteins of interest, but it also may be important to immortalize cellsimply so that they may be studied in vitro with greater ease. Inaddition, though many viruses provide promise as gene therapeuticvectors, these vectors may trigger apoptosis in the cells they infect.Blocking virally-induced apoptosis will prevent cell death caused bythese therapeutic vectors. As mentioned above, adenovirus, papillomaviruses, retrovirus, adeno-associated virus and HSV, for example, arecandidate gene therapeutic vectors that could benefit from thisapplication.

The general approach to inhibiting apoptosis, according to the presentinvention, will be to provide a cell with an U_(s)3 polypeptide, an ICP4polypeptide or both, thereby permitting the inhibitory activity ofU_(s)3, ICP4 or both to take effect. While it is conceivable that theprotein may be delivered directly, a preferred embodiment involvesproviding a nucleic acid encoding the polypeptide, i.e., an U_(s)3 gene,an α4 gene or both, to the cell. Following this provision, thepolypeptide is synthesized by the host cell's transcriptional andtranslational machinery, as well as any that may be provided by theexpression construct. Cis-acting regulatory elements necessary tosupport the expression of the U_(s)3 or α4 gene will be provided, asdescribed above, in the form of an expression construct. It also ispossible that, in the case of an HSV-infected cell, expression of thevirally-encoded U_(s)3 or ICP4 could be stimulated or enhanced, or theexpressed polypeptide stabilized, thereby achieving the same or similareffect.

In order to effect expression of constructs encoding U_(s)3 and/or ICPgenes, the expression construct must be delivered into a cell. Asdescribed above in the discussion of viral vectors, one mechanism fordelivery is via viral infection, where the expression construct isencapsidated in a viral particle which will deliver either a replicatingor non-replicating nucleic acid. The preferred embodiment is an HSVvector, although virtually any vector would suffice. Similarly, whereviral vectors are used to delivery other therapeutic genes, inclusion inthese vectors of an U_(s)3 and/or α4 gene advantageously will protectthe cell from virally induced apoptosis.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et. al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use, as discussed below.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro, but it may be applied toin vivo use as well. Another embodiment of the invention fortransferring a naked DNA expression construct into cells may involveparticle bombardment. This method depends on the ability to accelerateDNA coated microprojectiles to a high velocity allowing them to piercecell membranes and enter cells without killing them (Klein et al.,1987). Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold beads.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. In certainembodiments of the invention, the liposome may be complexed with ahemagglutinating virus (HVJ). This has been shown to facilitate fusionwith the cell membrane and promote cell entry of liposome-encapsulatedDNA (Kaneda et al., 1989). In other embodiments, the liposome may becomplexed or employed in conjunction with nuclear non-histonechromosomal proteins (HMG-1) (Kato et al., 1991). In yet furtherembodiments, the liposome may be complexed or employed in conjunctionwith both HVJ and HMG-1. In other embodiments, the delivery vehicle maycomprise a ligand and a liposome. Where a bacterial promoter is employedin the DNA construct, it also will be desirable to include within theliposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding an U_(s)3 and/or α4 transgene into cells arereceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis inalmost all eukaryotic cells. Because of the cell type-specificdistribution of various receptors, the delivery can be highly specific(Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994). Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties. In other embodiments, the delivery vehicle may comprise aligand and a liposome.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3,RIN and MDCK cells. In addition, a host cell strain may be chosen thatmodulates the expression of the inserted sequences, or modifies andprocess the gene product in the manner desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use—the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon (Phillips et al.,1985; Mizrahi, 1983). Cells are grown in a stainless steel tank with aheight-to-diameter ratio of 1:1 to 3:1. The culture is usually mixedwith one or more agitators, based on bladed disks or marine propellerpatterns. Agitator systems offering less shear forces than blades havebeen described. Agitation may be driven either directly or indirectly bymagnetically coupled drives. Indirect drives reduce the risk ofmicrobial contamination through seals on stirrer shafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gases and generatesrelatively low shear forces.

VII. Methods for the Induction of Apoptosis

In another embodiment of the present invention, there is contemplatedthe method of inducing apoptosis in HSV-infected cells, ie., blockingthe apoptotic function of US3, ICP4 or both. In this way, it may bepossible to curtail viral infection by bringing about a premature deathof the infected cell. In addition, it may prove effective to use thissort of therapeutic intervention in combination with more traditionalchemotherapies, such as the administration acyclovir.

The general form that this aspect of the invention will take is theprovision, to a cell, of an agent that will inhibit U_(s)3 function,ICP4 function or indeed inhibit the function of both. Four such agentsare contemplated as outlined below.

First, one may employ an antisense nucleic acid that will hybridizeeither to the U_(s)3 gene or the U_(s)3 transcript, thereby preventingtranscription or translation, respectively. Likewise, one may employ anantisense nucleic acid that will hybridize to an α4 gene or an α4transcript. The considerations relevant to the design of antisenseconstructs have been presented above.

Second, one may utilize an U_(s)3-binding protein or peptide, forexample, a peptidomimetic or an antibody that binds immunologically toan U_(s)3, the binding of either will block or reduce the activity of anU_(s)3. Again the same is true of ICP4 in that one may utilize anICP4-binding protein or peptide, for example, a peptidomimetic or anantibody that binds immunologically to an ICP4, the binding of eitherresulting in a block or reduction of ICP4 activity. The methods ofmaking and selecting peptide binding partners and antibodies are wellknown to those of skill in the art.

Third, one may provide to the cell an antagonist of U_(s)3 for example,the in the case of Us³ phosphorylation target sequence, alone or coupledto another agent. Equally one may provide to the cell an antagonist ofICP4, for example, the transactivation target sequence, alone or coupledto another agent. And fourth, one may provide an agent that binds to theU_(s)3 or ICP4 target without the same functional result as would arisewith U_(s)3 or ICP4 binding.

Provision of an U_(s)3 gene, an U_(s)3-binding protein, an U_(s)3antagonist, or an α4 gene, an ICP4-binding protein, or an ICP4antagonist, would be according to any appropriate pharmaceutical route.The formulation of such compositions and their delivery to tissues isdiscussed below. The method by which the nucleic acid, protein orchemical is transferred, along with the preferred delivery route, willbe selected based on the particular site to be treated. Those of skillin the art are capable of determining the most appropriate methods basedon the relevant clinical considerations.

Many of the gene transfer techniques that generally are applied in vitrocan be adapted for ex vivo or in vivo use. For example, selected organsincluding the liver, skin, and muscle tissue of rats and mice have beenbombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). Naked DNAalso has been used in clinical settings to effect gene therapy. Theseapproaches may require surgical exposure of the tumor tissue or directintratumoral injection. Nicolau et al. (1987) accomplished successfulliposome-mediated gene transfer in rats after intravenous injection.

Dubensky et al. (1984) successfully injected polyomavirus DNA in theform of CaPO₄ precipitates into liver and spleen of adult and newbornmice demonstrating active viral replication and acute infection.Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of CaPO₄ precipitated plasmids results inexpression of the transfected genes. Thus, it is envisioned that DNAencoding an antisense construct also may be transferred in a similarmanner in vivo.

Where the embodiment involves the use of an antibody that recognizes anU_(s)3 or an ICP4 polypeptide, consideration must be given to themechanism by which the antibody is introduced into the cell cytoplasm.This can be accomplished, for example, by providing an expressionconstruct that encodes a single-chain antibody version of the antibodyto be provided. Most of the discussion above relating to expressionconstructs for antisense versions of the respective genes will berelevant to this aspect of the invention. Alternatively, it is possibleto present a bifunctional antibody, where one antigen binding arm of theantibody recognizes an U_(s)3 or ICP4 polypeptide and the other antigenbinding arm recognizes a receptor on the surface of the cell to betargeted. Examples of suitable receptors would be an HSV glycoproteinsuch as gB, gC, gD, or gH. In addition, it may be possible to exploitthe Fc-binding function associated with HSV gE, thereby obviating theneed to sacrifice one arm of the antibody for purposes of celltargeting.

Advantageously, one may combine this approach with more conventionalchemotherapeutic options. Acyclovir is an active agent against HSV-1 andHSV-2. The drug inhibits actively replicating herpes virus but is notactive against latent virus. Acyclovir is available in threeformulations. For topical use, a five percent ointment producestherapeutic drug levels in mucocutaneous lesions. For systemic use,acyclovir may be administered orally or intravenously. The usualintravenous dosage in adults with normal renal function is 5 mg/kginfused at a constant rate over one hour and given every eight hours;this dosage produces peak plasma levels at about 10 g/ml. For HSVencephalitis, twice this dose is used. The usual adult oral dosage is200 mg, five times daily, which produces plasma levels that are lessthan 10% as high as those achieved with intravenous administration; eventhese levels are inhibitory to the virus, however. Acyclovir is given inan oral dosage of 800 mg five times daily for the treatment of herpeszoster, although oral administration generally is reserved for patientswith severe symptoms. A three percent opthalmic preparation producesinhibitory drug levels in the aqueous humor and is effective for herpeskeratitis.

In contemplating combinatorial aspects of the present invention, it willbe possible to combine the U_(s)3 therapeutic compositions of thepresent invention with other apoptosis inducing or inhibitingcompositions. For example the present inventors have show that it ispossible to affect apoptosis using compositions derived from ICP4protein or polypeptide (SEQ ID NO:2) and the a4 gene or gene constructsprotein or polypeptide (SEQ ID NO: 1). Thus in light of the teachingspresented herein, it will be possible for one of ordinary skill in theart to combine the U_(s)3 therapeutic compositions with those derivedfrom ICP4 and a4 to affect apoptosis as described herein below.

To kill cells, inhibit cell growth, or induce apoptosis as definedabove, using the methods and compositions of the present invention, onewould generally contact a “target” cell with a U_(s)3 and/or an ICP4expression construct alone or in combination with at least one otheragent. These compositions would be provided in a combined amounteffective to kill or induce apoptosis of the cell. This process mayinvolve contacting the cells with the expression construct and theagent(s) or factor(s) at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the expression construct and the other includes theagent.

Alternatively, the gene therapy treatment may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of eitherU_(s)3 or the other agent will be desired. Various combinations may beemployed, where U_(s)3 is “A” and the other agent is “B”, as exemplifiedbelow:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated where ICP4 and a4 may also be used.Again, to achieve apoptosis, both agents are delivered to a cell in acombined amount effective induce cell death.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventors propose that the regional delivery of U_(s)3 expressionconstructs and/or ICP4 expression constructs to patients with HSV linkeddisease will be a very efficient method for delivering a therapeuticallyeffective gene to counteract the clinical disease. Similarly, thechemotherapy may be directed to a particular, affected region of thesubjects body. Alternatively, systemic delivery of expression constructand/or the agent may be appropriate in certain circumstances, forexample, where extensive metastasis has occurred.

In addition to combining U_(s)3-targeted therapies with chemotherapies,it also is contemplated that combination with other gene therapies willbe advantageous. For example, targeting of U_(s)3 and α4 mutations atthe same time may produce an improved apoptotic treatment.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating a U_(s)3 related disease. In thisregard, reference to chemotherapeutics and non-U_(s)3 gene therapy incombination should also be read as a contemplation that these approachesmay be employed separately.

VIII. Methods for the Inhibition of Virus-Induced Cell Death In Vivo

In another embodiment of the present invention, there are providedmethods for the inhibition of cell death induced in vivo by any causecomprising the provision of U_(s)3 polypeptides or U_(s)3 genes, or ICP4polypeptides and α4 genes or combinations of the two. Also contemplatedin this aspect of the invention is the stimulation of viral U_(s)3expression, and/or stimulation of viral ICP4 expression or stabilizationof the virally-expressed U_(s)3 polypeptide and or ICP4 polypeptide.Though inhibition of apoptosis generally is thought of as advantageousto the virus, it may be desirable to effect this result as part of amethod of treating a viral infection. For example, if the host cellremains viable, the virus may continue to replicate; alternatively, ifapoptosis were occurring, the virus might be inclined to “go latent” inthe neural ganglia, where chemotherapeutic intervention is not helpful.Thus, by preventing early death of the cell, U_(s)3 and/or ICP4 maycause the virus to remain susceptible to treatment where it otherwisewould escape.

The mechanisms for delivering proteins and nucleic acids to a cell arediscussed elsewhere in this document and need not be repeated here. Theuse of standard chemotherapeutics has been presented in the precedingsection, and is incorporated in this section.

IX. Pharmaceuticals and In Vivo Methods for the Treatment of Disease

Aqueous pharmaceutical compositions of the present invention will havean effective amount of an U_(s)3 and/or a4 expression construct, anantisense U_(s)3 and/or α4 expression construct, an expression constructthat encodes a therapeutic gene along with U_(s)3 and/or α4, a proteinthat inhibits U_(s)3 and/or α4 function, such as an anti-US3 antibody oran anti-ICP4 antibody, respectively, or an U_(s)3 polypeptide and/or anICP4 polypeptide. Such compositions generally will be dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium. An“effective amount,” for the purposes of therapy, is defined at thatamount that causes a clinically measurable difference in the conditionof the subject. This amount will vary depending on the substance, thecondition of the patient, the type of treatment, the location of thelesion, etc.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, orhuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredients,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients, such as other anti-cancer agents, can also beincorporated into the compositions.

In addition to the compounds formulated for parenteral administration,such as those for intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g., tablets or other solidsfor oral administration; time release capsules; and any other formcurrently used, including cremes, lotions, mouthwashes, inhalants andthe like.

The active compounds of the present invention will often be formulatedfor parenteral administration, e.g., formulated for injection via theintravenous, intramuscular, subcutaneous, or even intraperitonealroutes. The preparation of an aqueous composition that containsglycosylceramide synthesis inhibitory compounds alone or in combinationwith a chemotherapeutic agent as active ingredients will be known tothose of skill in the art in light of the present disclosure. Typically,such compositions can be prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for using to preparesolutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and the preparations can also beemulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In many cases, the form must be sterile and must be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

The active compounds may be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylarnine, histidine, procaine and thelike.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In certain cases, the therapeutic formulations of the invention couldalso be prepared in forms suitable for topical administration, such asin cremes and lotions. These forms may be used for treatingskin-associated diseases, such as various sarcomas. In certain othercases, the formulation will be geared for administration to the centralnervous system, e.g., the brain.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,with even drug release capsules and the like being employable.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

X. Examples

The following examples are included to demonstrate preferred embodimentsof the present invention. It should be appreciated by those of skill inthe art that that techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar results without departing from the spirit andscope of the invention.

EXAMPLE 1

Materials And Methods

Cells and Viruses. Vero and BHK cells were originally obtained fromATCC. HSV-1(F) is the prototype HSV-1 strain used in this laboratory(Ejercito et al., 1967). In recombinant R325 derived from HSV-1(F),approximately 800 bp comprising the carboxyl-terminal half of the α4gene and grows only in a Vero cell line expressing the α4 gene (DeLucaet al., 1985). Both the virus and the cell line were kind gifts of NealDeLuca (University of Pittsburgh). The U_(s)3 gene was deleted from therecombinant R7041 (Longnecker et al., 1987) and then repaired to yieldR7306).

Plasmids and Cosmids. The plasmid pRB5166 was constructed by cloninginto the SaII/BamHI sites of the pACYC184 vector a HSV-1 DNA fragmentthat extends from the Sal I site at +177 with respect to the α4 genetranscription start site and includes the entire BamHI P and BamHI Sfragments. The cosmid cloning vector pRB78 was constructed as follows.The multiple cloning site of the STRAGENE®SUPERCOSL® (La Jolla Calif.,cat#251301) was cleaved with EcoRI and replaced with an oligonucleotidescontaining EcoRI/PacI/Sse8387I/SpeI/BarnHI/NdeI/EcoRV/PacI/EcoRI cloningsites. The PacI restriction site, absent in HSV-1, serves to liberatesthe cloned HSV-1(F) DNA fragments from the vector. HSV-1(F) viral DNAwas prepared from virions as previously described. To construct cosmidpBC1004, which contains the HSV-1(F) sequence n133052 through n17059,viral DNA was partially digested with Sau3A I, dephosphorylated andligated into the BamHI site of the pRB78 cosmid vector previouslylinearized with Xba I. The DNA was then packaged using STRAGENE GIGAPACKXLII ® (La Jolla, Calif.), following the manufacturer's instructions. E.coli L-1 Blue MR was then infected and ampicillin-resistant colonieswere screened by restriction enzyme analysis. Insert termini weresequenced to verify mapping. Cosmid pBC1008 was constructed by cloningthe BglII F-H fragment (HSV-1(F) n106750 through n142759) into the BamHIsite of pRB78 vector. Cosmid pBC1009 was constructed as follows. AnNsiI/ScaI DNA fragment was isolated from a double digest of viral DNA.The fragment (HSV-1(F) n137538 through n18545) was ligated into theSse8387IlEcoRV sites of pBR78. Cosmids PBC1008 and PBC1009 were mappedby restriction enzyme analysis and insert termini were sequenced (FIG.1).

Electron photomicroscopy. Vero cells infected with HSV-1(F) or d120 wereincubated for 20 hrs at 37° C. Cells were fixed in 2% glutaraldehyde inPBS for 60 min at 4° C., post-fixed with 1% osmium tetroxide, en blocstained with uranyl acetate (5mg/ml), dehydrated in acetone, andembedded in EPON® 812. Thin sections were examined either unstained orpoststained with uranyl acetate and lead hydroxide. The cells werephotographed at 6000 X in a SIEMENS 101 electron microscope.

Light photomicroscopy. Cells were labeled with biotinylated dUTP. Atindicated times the cells were fixed in ice-cold methanol at −20° C. andair-dried, then rinsed in phosphate buffered saline, reacted for 15 minat room temperature with 40 ml of a solution containing 1× terminaltransferase buffer (PROMEGA®), 1 mM CoCl₂, 0.05 mg/ml bovine serumalbumin (BSA), 0.5 nmoles biotin-16-dUTP (BOEHRINGER MANNHEIMBIOCHEMICALS®) and 3 units of terminal transferase (TdT, PROMEGA),rinsed extensively in PBS, reacted for 30 min at room temperature with40 ml of a solution containing Texas red-conjugated avidin in 4× SSC (1×SSC is 0.15 M NaCl, 0.015 M Na-citrate), 0.1% Triton X-100, 5% (w/v)nonfat dry milk, extensively rinsed with PBS, mounted in 10% PBS inglycerol and examined under a ZEISS® confocal fluorescence microscope.The images were captured under identical settings with the softwareprovided by ZEISS® and printed in a TEKTRONIX® 400 phaser printer. Cellswere mock infected, 37° C, 20 hrs; infected with d120 virus, 37° C. 20hrs; infected with HSV-1(F), 37° C., 20 hrs; infected with d120 virus,37° C. 30 hrs; mock-unfected, 39.5° C., 30 hrs.; infected with d120virus, 39.5° C., 30 hrs.; and infected with HSV-1(F), 39.5° C., 30 hrs.

DNA Fragmentation Assay. Vero cells or E5 cells were infected withHSV-1(F), d120 mutant, or HSV-1 tsHA1 mutant and maintained at 37° C. or39.5° C. in the absence or in the presence of phosphonoacetic acid. At30 hrs after infection, 2×10⁶ cells per sample were collected, washed inPBS, lysed in a solution containing 10 mM Tris-HCl, pH 8.0, 10 mM EDTA,and 0.5 % Triton X-100, and centrifuged at 12,000 rpm for 25 min in anEppendorf microcentrifuge to pellet chromosomal DNA. Supernatant fluidswere digested with 0.1 mg RNase A per ml at 37° C. for 1 hr, for 2 hrswith 1 mg proteinase K per ml at 50° C. in the presence of 1% sodiumdodecylsulphate (SDS), extracted with phenol and chloroform, andprecipitated in cold ethanol and subjected to electrophoresis onhorizontal 1.5% agarose gels containing 5 mg of ethidium bromide per ml.DNA was visualized by UV light transillumination. Photographs were takenwith the aid of a computer-assisted image processor (Eagle Eye II,Stratagene).

Construction of recombinant viruses by marker rescue. Recombinantviruses were constructed using a modification of the techniqueoriginally described by Post and Roizman (1981). Vero cells weretransfected with plasmid or cosmid DNA using Lipofectamin, according tothe manufacturer's instructions. At 6 hr after transfection, the cellswere exposed to 0.1 to 1 PFU of HSV-1(Kos)d120 per cell. Recombinantviruses were isolated from single plaques and grown in Vero cells. Toobtain HSV-1 120KR, Vero cells were exposed to 10 PFU of HSV-1(KOS)d120per cell. The infected cells were harvested at 48 hr post infection,frozen-thawn and sonicated, and then serially diluted and titered onVero cells. Recombinant viruses were isolated from single plaques andgrown in Vero cells.

EXAMPLE 2

An HSV-1 mutant deleted in ICP4 induces apoptosis.

In this series of studies, Vero cells infected with wild-type or thed120 mutant were examined for morphologic evidence of apoptosis. Verocells were fixed and harvested at 20 to 24 hrs after infection withwild-type or d120, embedded, sectioned, and examined in a Siemens 101electron microscope. The cells infected with wild-type virus showedtypical infected cell morphology, ie., marginated chromatin, separationof inner and outer nuclear membranes, and accumulation of virusparticles in some but not all cells. Cells infected with the d120deletion mutant exhibited extensive condensation of chromatin,obliteration of the nuclear membrane, and extensive vacuolization andblebbing of the cytoplasm. It was estimated that approximately 40 to 50%of the infected cells exhibited some or all of the morphologic changesdescribed above.

In another series of studies, Vero cells were mock-infected or infectedwith 10 PFU of either the wild-type or the d120 mutant virus per cell.After 20 hrs of incubation at 37° C. the cells were fixed, labeled withbiotinylated dUTP in the presence of terminal transferase, and thenreacted with fluorescent avidin. Mock-infected cells or cells infectedwith wild-type virus showed no sign of labeling with biotinylated dUTPby terminal transferase, whereas cells infected with d120 and maintainedat the same temperature showed extensive fluorescence due to thereaction of fluorescent avidin to biotinylated dUTP incorporated at theDNA termini created by the cleavage of DNA.

In the third series of studies, replicate Vero cell cultures wereinfected with 10 PFU of either HSV-1(F) or d120 per cell and incubatedat 37° C. The study also included a Vero cell culture infected withHSV-1(F), overlaid with medium containing 300 μg of phosphonoacetate perml and incubated at 37° C., and a set of Vero cell cultures infectedwith 10 PFU of HSV-1 tsHA1 and incubated at either 37° C. or 39.5° C.This concentration of phosphonoacetate completely inhibits viral DNAsynthesis and blocks the expression of 72 genes dependent on viralsynthesis for their expression. The cells were harvested at 30 hrs afterinfection, lysed, and centrifuged to pellet the chromosomal DNA. Thesupernatant fluids were processed as described above and subjected toelectrophoresis in agarose gels to test for the presence of soluble,fragmented DNA.

The results were as follows. Cells infected with d120 deletion mutantyielded high amounts of fragmented DNA which were readily visible onagarose gels stained with ethidium bromide. These ladders were not seenin agarose gels containing electrophoretically separated extracts ofwild-type infected cells or E5 cells infected with d120. When Vero cellswere incubated in medium containing phosphonoacetate, fragmented DNA wasdetected from cells infected with d120 mutant but not with thewild-type. Fragmented DNA was visible in extracts of mock-infected cellsincubated at 39.5° C., but not in cells infected with HSV-1 tsHA1 andincubated at the same temperature.

From this series of studies, it is concluded that (i) HSV-1 is capableof inducing the morphologic and biochemical changes characteristic ofapoptosis and these changes are prominent in cells infected with amutant lacking ICP4; (ii) wild-type virus does not induce apoptosisindicating that ICP4 or a protein expressed subsequently is able toprotect cells from apoptosis; (iii) the protective, anti-apoptoticeffect is a viral function which does not depend on the onset of viralDNA synthesis; (iv) DNA degradation typical of apoptosis was observedupon incubation at 39.5 ° C. in mock-infected but not HSV-1tsHA1infected cells, which suggests that prolonged incubation at the elevatedtemperature can induce apoptosis that is blocked by a viral functionexpressed early.

EXAMPLE 3

ICP4 expresses an anti-apoptotic function.

In a fourth series of studies, Vero or E5 cells were mock infected orinfected with 10 PFU per cell with either HSV-1(F) or d120. The cellswere incubated at 39.5° C. for 30 hrs. The rationale of these studieswas as follows. As noted in Example 1, HSV-1(F) carries a ts lesion inthe a4 gene and at the nonpermissive temperature (39.5° C.) expressesonly α genes. The α4 gene resident in the E5 cell line and the d120mutant virus lacking the α4 gene were derived from the HSV-1(KOS) strainwhich does not exhibit the ts phenotype. In addition, the α4 generesident in the E5 cell line is induced after infection and is notexpressed in uninfected cells. In the first series of studies, the cellswere harvested, lysed, centrifuged to sediment chromosomal DNA and thesupernatant fluids were processed as described in Example 1 andsubjected to electrophoresis in agarose gels.

The results were as follows. Fragmented DNA was present in lanescontaining electrophoretically separated extracts of mock-infected Verocells, Vero cells infected with d120 mutant, and the mock infected E5cells. Fragmented DNA was not detected in Vero cells infected withwild-type virus, or in E5 cells infected with either d120 mutant virusor HSV-1(F) virus.

In the second series of studies Vero cells were mock infected, orinfected with either d120 or with wild-type virus. After 30 hrs ofincubation at 39.5° C, the cells were fixed and labeled withbiotinylated dUTP by terminal transferase, and reacted with fluorescentavidin. Fluorescence was detected in mock-infected or infected with d120mutant, but not in cells infected with wild-type virus.

These studies allowed permit the conclusion conclude that HSV-1(F) α4gene encodes a function which blocks apoptosis reflected in thedegradation of DNA, and that this function is separable from therepressor and transactivator functions of ICP4 which are affected by thetemperature sensitive lesion of the α4 gene of HSV-1 (F).

A summary of the results is provided in Table 4. Induction of (+), orprotection from (−) apoptosis is indicated upon conditions (infectingvirus and incubation temperature) which induce apoptosis in Vero and E5cell lines. “nt” indicates not tested.

TABLE 4 VERO VERO (37° C.) (39° C.) E5 (37° C.) E5 (39.5° C.) MOCK − +− + HSV-1(F) − − − − HSV-1 d120 + + − − HSV-1 tsHA1 − − nt −

EXAMPLE 4

HSV-1(KOS)d120 carries an additional mutation.

The results described in this section emerged from studies designed torepair the deletion in both copies of the α4 gene of HSV-1(KOS)d120. Theinventors isolated recombinant in which this gene was repaired by twodifferent procedures. In the first, the inventors cloned an HSV-1(F) DNAfragment that contains an α4 sequence plus enough flanking sequence toallow homologous recombination. In this series of studies Vero cellswere transfected with plasmid DNA and infected with 0.1 to 1 PFU ofHSV-1(KOS)d120 per cell. Under these conditions, plaques formed only incultures of cells transfected with the plasmid DNA. Recombinant viruswas recovered from individual plaques, and designated 120FR [forHSV-1(F) repair].

The second procedure was based on the observation that a small amount ofvirus recombines with the resident α4 gene in the E5 cell line to yieldrescued virus capable of replicating efficiently in the absence ofexogenous source of ICP4. The observed recombination frequency is 10⁻⁶to 10⁻⁷ and therefore such rescued virus would be expected to be presentin HSV-1(KOS)d120 stock. To isolate these recombinants, Vero cells wereinfected at a high multiplicity with HSV-1 d120. At 24 hr afterinfection cells were harvested, frozen-thawed and serial dilutions wereused to infect Vero cells. The recombinant virus obtained in thisfashion was designated 120KR [for HSV-1(KOS) repair].

As shown in FIG. 3, analyses of DNA extracted from replicate Vero cellcultures infected with HSV-1(KOS)d120, 120FR or 120KR showed ladderstypical of apopotic cells. These ladders were absent in extracts ofmock-infected cells or cells infected with HSV-1(F). The inventorsconclude from these studies that HSV-1(KOS)d120 genome contain anadditional mutation other than in the α4 gene.

EXAMPLE 5

The U_(s)3 protein kinase is required to block apoptosis induced byHSV-1 infection.

In the following series of studies, the inventors defined the regionthat contains the additional mutation in HSV-1(KOS)d120 by rescue of theHSV-1(KOS)d120 with cosmids containing large HSV-1 DNA fragments. Thethree cosmids used in these studies were cosmid pBC8008, which containsall of the HSV-1 terminal repeat sequence, almost all of the U_(s)sequence, and part of the U_(L) sequence (FIG. 1) and cosmids pBC8004and pBC8009 which contain fragments spanning over the entire repeatsequence but differ in the extent of coverage of the U_(s) region.Individual plaques purified from each transfection were tested for theirability to protect infected cells from apoptosis induced by infection.The results shown in Table 5 suggested that the second mutation inHSV-1(KOS)d120 may map in the U_(s) domain containing the genes U_(s)1through U_(s)3.

TABLE 5 Plasmid/Cosmid Recombinant Protection From Apoptosis pRB5166HSV-1 120FR 0/9 none HSV-1 120KR 0/5 pBC1004 HSV-1 120AR 1/4 pBC1009HSV-1 120BR 0/9 pBC1008 HSV-1 120CR 8/9

To map the function required to block apoptosis more precisely, theinventors took advantage of the availability in this laboratory ofseveral deletion mutants which span the sequence thought to encode thegene required to block apoptosis in infected cells. Recombinant virusR325 lacks the carboxyl terminal half of the α22 gene, and the 3′ domainof the U_(s)2 gene. Recombinant virus R7041, lacks most of the U_(s)3gene whereas in recombinant R7306 the U_(s)3 gene had been repaired. Theresults, shown in FIG. 3 indicate that apoptosis was induced in cellsinfected with R7041 (ΔU_(s)3) but not in cells infected with the othermutants.

Two series of studies verified the conclusion that a functional U_(s)3gene is required for prevention of apoptosis and that the secondmutation in the HSV-1(KOS)d120 resides in the U_(s)3 gene. In the firstseries, the inventors carried out simple complementation tests todetermine whether the second mutation in HSV-1(KOS)d120 was the same asthat in the rescued virus or in R7041 (ΔU_(s)3) recombinant.Specifically Vero cells were infected with artificial mixtures of 120FRand HSV-1(KOS)d120, 120FR and R7041, or 120FR and R7306 (U_(s)3repaired). The results, shown in FIG. 4 indicate the following. As couldbe expected, 120FR did not complement HSV-1(KOS)d120, the parent virusfrom which it was derived. The second, mixture of 120FR and R7041 alsodid not complement each other suggesting that 120FR and its parentvirus, HSV-1(KOS)d120 contained a nonfunctional U_(s)3. Verification ofthis hypothesis emerged from the observation that R7306 containing arepaired U_(s)3 gene complemented 120FR and blocked apoptosis in cellsinfected with these two viruses.

The second series of studies served to verify the HSV-1(KOS)d120 and itsderivatives lacked a functional U_(s)3 gene. As described in thisintroduction, the production of U_(L)34 gene has been shown in earlierstudies from this laboratory to be a substrate for the U_(s)3 proteinkinase. In wild type infected cells, U_(L) ³4 has an apparent M_(r) of30,000 whereas in cells infected with R7041 (AU_(s)3), U_(L)3⁴ has anapparent migrates of M_(r) 33,000. Inasmuch as the original studies weredone in BHK cells, in this series of studies replicate cultures of BHKcells were infected 10 PFU of HSV-1(F), 120FR, R7041, and R7306 percell. The results, shown in FIG. 5 indicate that the fully processedU_(L)34 protein was present in cells infected with HSV-1(F) but notcells infected with 120FR or R7041. As could be expected, the cellsinfected with HSV-1(KOS)d120 did not make detectable quantities ofU_(L)34 protein inasmuch as they lack the a4 gene. In the absense of α4gene expression there is no β or γ gene expression and therefore, noU_(s)3 gene expression. Taken together, the inventors' results indicatethe U_(s)3 kinase is functionally absent in HSV-1(KOS)d120 and 120FR.The inventors conclude that U_(s)3 is required for protection fromapoptosis induced by HSV-1.

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6 4257 base pairs nucleic acid single linear unknown 1 GCTGCTCCTCCTTCCCGCCG GCCCCTGGGA CTATATGAGC CCGAGGACGC CCCGATCGTC 60 CACACGGAGCGCGGCTGCCG ACACGGATCC ACGACCCGAC GCGGGACCGC CAGAGACAGA 120 CCGTCAGACGCTCGCCGCGC CGGGACGCCG ATACGCGGAC GAAGCGCGGG AGGGGGATCG 180 GCCGTCCCTGTCCTTTTTCC CACCCAAGCA TCGACCGGTC CGCGCTAGTT CCGCGTCGAC 240 GGCGGGGGTCGTCGGGGTCC GTGGGTCTCG CCCCCTCCCC CCATCGAGAG TCCGTAGGTG 300 ACCTACCGTGCTACGTCCGC CGTCGCAGCC GTATCCCCGG AGGATCGCCC CGCATCGGCG 360 ATGGCGTCGGAGAACAAGCA GCGCCCCGGC TCCCCGGGCC CCACCGACGG GCCGCCGCCC 420 ACCCCGAGCCCAGACCGCGA CGAGCGGGGG GCCCTCGGGT GGGGCGCGGA GACGGAGGAG 480 GGCGGGGACGACCCCGACCA CGACCCCGAC CACCCCCACG ACCTCGACGA CGCCCGGCGG 540 GACGGGAGGGCCCCCGCGGC GGGCACCGAC GCCGGCGAGG ACGCCGGGGA CGCCGTCTCG 600 CCGCGACAGCTGGCTCTGCT GGCCTCCATG GTAGAGGAGG CCGTCCGGAC GATCCCGACG 660 CCCGACCCCGCGGCCTCGCC GCCCCGGACC CCCGCCTTTC GAGCCGACGA CGATGACGGG 720 GACGAGTACGACGACGCAGC CGACGCCGCC GGCGACCGGG CCCCGGCCCG GGGCCGCGAA 780 CGGGAGGCCCCGCTACGCGG CGCGTATCCG GACCCCACGG ACCGCCTGTC GCCGCGCCCG 840 CCGGCCCAGCCGCCGCGGAG ACGTCGTCAC GGCCGGTGGC GGCCATCGGC GTCATCGACC 900 TCGTCGGACTCCGGGTCCTC GTCCTCGTCG TCCGCATCCT CTTCGTCCTC GTCGTCCGAC 960 GAGGACGAGGACGACGACGG CAACGACGCG GCCGACCACG CACGCGAGGC GCGGGCCGTC 1020 GGGCGGGGTCCGTCGAGCGC GGCGCCGGCA GCCCCCGGGC GGACGCCGCC CCCGCCCGGG 1080 CCACCCCCCCTCTCCGAGGC CGCGCCCAAG CCCCGGGCGG CGGCGAGGAC CCCCGCGGCC 1140 TCCGCGGGCCGCATCGAGCG CCGCCGGGCC CGCGCGGCGG TGGCCGGCCG CGACGCCACG 1200 GGCCGCTTCACGGCCGGGCA GCCCCGGCGG GTCGAGCTGG ACGCCGACGC GACCTCCGGC 1260 GCCTTCTACGCGCGCTATCG CGACGGGTAC GTCAGCGGGG AGCCGTGGCC CGGCGCCGGG 1320 CCCCCGCCCCCGGGGCGGGT GCTGTACGGC GGCCTGGGCG ACAGCCGCCC GGGCCTCTGG 1380 GGGGCGCCCGAGGCGGAGGA GGCGCGACGC CGGTTCGAGG CCTCGGGCGC CCCGGCGGCC 1440 GTGTGGGCGCCCGAGCTGGG CGACGCCGCG CAGCAGTACG CCCTGATCAC GCGGCTGCTG 1500 TACACCCCGGACGCGGAGGC CATGGGGTGG CTCCAGAACC CGCGCGTGGT CCCCGGGGAC 1560 GTGGCGCTGGACCAGGCCTG CTTCCGGATC TCGGGCGCCG CGCGCAACAG CAGCTCCTTC 1620 ATCACCGGCAGCGTGGCGCG GGCCGTGCCC CACCTGGGCT ACGCCATGGC GGCCGGCCGC 1680 TTCGGCTGGGGCCTGGCGCA CGCGGCGGCC GCCGTGGCCA TGAGCCGCCG ATACGACCGC 1740 GCGCAGAAGGGCTTCCTGCT GACCAGCCTG CGCCGCGCCT ACGCGCCCCT GTTGGCGCGC 1800 GAGAACGCGGCGCTGACGGG GGCCGCGGGG AGCCCCGGCG CCGGCGCAGA TGACGAGGGG 1860 GTCGCCGCCGTCGCCGCCGC CGCACCGGGC GAGCGCGCGG TGCCCGCCGG GTACGGCGCC 1920 GCGGGGATCCTCGCCGCCCT GGGGCGGCTG TCCGCCGCGC CCGCCTCCCC CGCGGGGGGC 1980 GACGACCCCGACGCCGCCCG CCACGCCGAC GCCGACGACG ACGCCGGGCG CCGCGCCCAG 2040 GCCGGCCGCGTGGCCGTCGA GTGCCTGGCC GCCTGCCGCG GGATCCTGGA GGCGCTGGCC 2100 GAGGGCTTCGACGGCGACCT GGCGGCCGTC CCGGGGCTGG CCGGGGCCCG GCCCGCCAGC 2160 CCCCCGCGGCCGGAGGGACC CGCGGGCCCC GCTTCCCCGC CGCCGCCGCA CGCCGACGCG 2220 CCCCGCCTGCGCGCGTGGCT GCGCGAGCTG CGGTTCGTGC GCGACGCGCT GGTGCTCATG 2280 CGCCTGCGCGGGGACCTGCG CGTGGCCGGC GGCAGCGAGG CCGCCGTGGC CGCCGTGCGC 2340 GCCGTGAGCCTGGTCGCCGG GGCCCTGGGC CCCGCGCTGC CGCGGGACCC GCGCCTGCCG 2400 AGCTCCGCGGCCGCCGCCGC CGCGGACCTG CTGTTTGACA ACCAGAGCCT GCGCCCCCTG 2460 CTGGCGGCGGCGGCCAGCGC ACCGGACGCC GCCGACGCGC TGGCGGCCGC CGCCGCCTCC 2520 GCCGCGCCGCGGGAGGGGCG CAAGCGCAAG AGTCCCGGCC CGGCCCGGCC GCCCGGAGGC 2580 GGCGGCCCGCGACCCCCGAA GACGAAGAAG AGCGGCGCGG ACGCCCCCGG CTCGGACGCC 2640 CGCGCCCCCCTCCCCGCGCC CGCGCCCCCC TCCACGCCCC CGGGGCCCGA GCCCGCCCCC 2700 GCCCAGCCCGCGGCGCCCCG GGCCGCCGCG GCGCAGGCCC GCCCGCGCCC CGTGGCCGTG 2760 TCGCGCCGGCCCGCCGAGGG CCCCGACCCC CTGGGCGGCT GGCGGCGGCA GCCCCCGGGG 2820 CCCAGCCACACGGCGGCGCC CGCGGCCGCC GCCCTGGAGG CCTACTGCTC CCCGCGCGCC 2880 GTGGCCGAGCTCACGGACCA CCCGCTGTTC CCCGTCCCCT GGCGACCGGC CCTCATGTTT 2940 GACCCGCGGGCCCTGGCCTC GATCGCCGCG CGGTGCGCCG GGCCCGCCCC CGCCGCCCAG 3000 GCCGCGTGCGGCGGCGGCGA CGACGACGAT AACCCCCACC CCCACGGGGC CGCCGGGGGC 3060 CGCCTCTTTGGCCCCCTGCG CGCCTCGGGC CCGCTGCGCC GCATGGCGGC CTGGATGCGC 3120 CAGATCCCCGACCCCGAGGA CGTGCGCGTG GTGGTGCTGT ACTCGCCGCT GCCGGGCGAG 3180 GACCTGGCCGGCGGCGGGGC CTCGGGGGGG CCGCCGGAGT GGTCCGCCGA GCGCGGCGGG 3240 CTGTCCTGCCTGCTGGCGGC CCTGGCCAAC CGGCTGTGCG GGCCGGACAC GGCCGCCTGG 3300 GCGGGCAATTGGACCGGCGC CCCCGACGTG TCGGCGCTGG GCGCACAGGG CGTGCTGCTG 3360 CTGTCCACGCGGGACCTGGC CTTCGCCGGG GCCGTGGAGT TTCTGGGGCT GCTCGCCAGC 3420 GCCGGCGACCGGCGGCTCAT CGTGGTCAAC ACCGTGCGCG CCTGCGACTG GCCCGCCGAC 3480 GGGCCCGCGGTGTCGCGGCA GCACGCCTAC CTGGCGTGCG AGCTGCTGCC CGCCGTGCAG 3540 TGCGCCGTGCGCTGGCCGGC GGCGCGGGAC CTGCGCCGCA CGGTGCTGGC CTCGGGCCGC 3600 GTGTTCGGCCCGGGGGTCTT CGCGCGCGTG GAGGCCGCGC ACGCGCGCCT GTACCCCGAC 3660 GCGCCGCCGCTGCGCCTGTG CCGCGGCGGC AACGTGCGCT ACCGCGTGCG CACGCGCTTC 3720 GGCCCGGACACGCCGGTGCC CATGTCCCCG CGCGAGTACC GCCGGGCCGT GCTGCCGGCG 3780 CTGGACGGCCGGGCGGCGGC CTCGGGGACC ACCGACGCCA TGGCGCCCGG CGCGCCGGAC 3840 TTCTGCGAGGAGGAGGCCCA CTCGCACGCC GCCTGCGCGC GCTGGGGCCT GGGCGCGCCG 3900 CTGCGGCCCGTGTACGTGGC GCTGGGGCGC GAGGCGGTGC GCGCCGGCCC GGCCCGGTGG 3960 CGCGGGCCGCGGAGGGACTT TTGCGCCCGC GCCCTGCTGG AGCCCGACGA CGACGCCCCC 4020 CCGCTGGTGCTGCGCGGCGA CGACGACGGC CCGGGGGCCC TGCCGCCGGC GCCGCCCGGG 4080 ATTCGCTGGGCCTCGGCCAC GGGCCGCAGC GGCACCGTGC TGGCGGCGGC GGGGGCCGTG 4140 GAGGTGCTGGGGGCGGAGGC GGGCTTGGCC ACGCCCCCGC GGCGGGAAGT TGTGGACTGG 4200 GAAGGCGCCTGGGACGAAGA CGACGGCGGC GCGTTCGAGG GGGACGGGGT GCTGTAA 4257 1298 aminoacids amino acid linear unknown 2 Met Ala Ser Glu Asn Lys Gln Arg ProGly Ser Pro Gly Pro Thr Asp 1 5 10 15 Gly Pro Pro Pro Thr Pro Ser ProAsp Arg Asp Glu Arg Gly Ala Leu 20 25 30 Gly Trp Gly Ala Glu Thr Glu GluGly Gly Asp Asp Pro Asp His Asp 35 40 45 Pro Asp His Pro His Asp Leu AspAsp Ala Arg Arg Asp Gly Arg Ala 50 55 60 Pro Ala Ala Gly Thr Asp Ala GlyGlu Asp Ala Gly Asp Ala Val Ser 65 70 75 80 Pro Arg Gln Leu Ala Leu LeuAla Ser Met Val Glu Glu Ala Val Arg 85 90 95 Thr Ile Pro Thr Pro Asp ProAla Ala Ser Pro Pro Arg Thr Pro Ala 100 105 110 Phe Arg Ala Asp Asp AspAsp Gly Asp Glu Tyr Asp Asp Ala Ala Asp 115 120 125 Ala Ala Gly Asp ArgAla Pro Ala Arg Gly Arg Glu Arg Glu Ala Pro 130 135 140 Leu Arg Gly AlaTyr Pro Asp Pro Thr Asp Arg Leu Ser Pro Arg Pro 145 150 155 160 Pro AlaGln Pro Pro Arg Arg Arg Arg His Gly Arg Trp Arg Pro Ser 165 170 175 AlaSer Ser Thr Ser Ser Asp Ser Gly Ser Ser Ser Ser Ser Ser Ala 180 185 190Ser Ser Ser Ser Ser Ser Ser Asp Glu Asp Glu Asp Asp Asp Gly Asn 195 200205 Asp Ala Ala Asp His Ala Arg Glu Ala Arg Ala Val Gly Arg Gly Pro 210215 220 Ser Ser Ala Ala Pro Ala Ala Pro Gly Arg Thr Pro Pro Pro Pro Gly225 230 235 240 Pro Pro Pro Leu Ser Glu Ala Ala Pro Lys Pro Arg Ala AlaAla Arg 245 250 255 Thr Pro Ala Ala Ser Ala Gly Arg Ile Glu Arg Arg ArgAla Arg Ala 260 265 270 Ala Val Ala Gly Arg Asp Ala Thr Gly Arg Phe ThrAla Gly Gln Pro 275 280 285 Arg Arg Val Glu Leu Asp Ala Asp Ala Thr SerGly Ala Phe Tyr Ala 290 295 300 Arg Tyr Arg Asp Gly Tyr Val Ser Gly GluPro Trp Pro Gly Ala Gly 305 310 315 320 Pro Pro Pro Pro Gly Arg Val LeuTyr Gly Gly Leu Gly Asp Ser Arg 325 330 335 Pro Gly Leu Trp Gly Ala ProGlu Ala Glu Glu Ala Arg Arg Arg Phe 340 345 350 Glu Ala Ser Gly Ala ProAla Ala Val Trp Ala Pro Glu Leu Gly Asp 355 360 365 Ala Ala Gln Gln TyrAla Leu Ile Thr Arg Leu Leu Tyr Thr Pro Asp 370 375 380 Ala Glu Ala MetGly Trp Leu Gln Asn Pro Arg Val Val Pro Gly Asp 385 390 395 400 Val AlaLeu Asp Gln Ala Cys Phe Arg Ile Ser Gly Ala Ala Arg Asn 405 410 415 SerSer Ser Phe Ile Thr Gly Ser Val Ala Arg Ala Val Pro His Leu 420 425 430Gly Tyr Ala Met Ala Ala Gly Arg Phe Gly Trp Gly Leu Ala His Ala 435 440445 Ala Ala Ala Val Ala Met Ser Arg Arg Tyr Asp Arg Ala Gln Lys Gly 450455 460 Phe Leu Leu Thr Ser Leu Arg Arg Ala Tyr Ala Pro Leu Leu Ala Arg465 470 475 480 Glu Asn Ala Ala Leu Thr Gly Ala Ala Gly Ser Pro Gly AlaGly Ala 485 490 495 Asp Asp Glu Gly Val Ala Ala Val Ala Ala Ala Ala ProGly Glu Arg 500 505 510 Ala Val Pro Ala Gly Tyr Gly Ala Ala Gly Ile LeuAla Ala Leu Gly 515 520 525 Arg Leu Ser Ala Ala Pro Ala Ser Pro Ala GlyGly Asp Asp Pro Asp 530 535 540 Ala Ala Arg His Ala Asp Ala Asp Asp AspAla Gly Arg Arg Ala Gln 545 550 555 560 Ala Gly Arg Val Ala Val Glu CysLeu Ala Ala Cys Arg Gly Ile Leu 565 570 575 Glu Ala Leu Ala Glu Gly PheAsp Gly Asp Leu Ala Ala Val Pro Gly 580 585 590 Leu Ala Gly Ala Arg ProAla Ser Pro Pro Arg Pro Glu Gly Pro Ala 595 600 605 Gly Pro Ala Ser ProPro Pro Pro His Ala Asp Ala Pro Arg Leu Arg 610 615 620 Ala Trp Leu ArgGlu Leu Arg Phe Val Arg Asp Ala Leu Val Leu Met 625 630 635 640 Arg LeuArg Gly Asp Leu Arg Val Ala Gly Gly Ser Glu Ala Ala Val 645 650 655 AlaAla Val Arg Ala Val Ser Leu Val Ala Gly Ala Leu Gly Pro Ala 660 665 670Leu Pro Arg Asp Pro Arg Leu Pro Ser Ser Ala Ala Ala Ala Ala Ala 675 680685 Asp Leu Leu Phe Asp Asn Gln Ser Leu Arg Pro Leu Leu Ala Ala Ala 690695 700 Ala Ser Ala Pro Asp Ala Ala Asp Ala Leu Ala Ala Ala Ala Ala Ser705 710 715 720 Ala Ala Pro Arg Glu Gly Arg Lys Arg Lys Ser Pro Gly ProAla Arg 725 730 735 Pro Pro Gly Gly Gly Gly Pro Arg Pro Pro Lys Thr LysLys Ser Gly 740 745 750 Ala Asp Ala Pro Gly Ser Asp Ala Arg Ala Pro LeuPro Ala Pro Ala 755 760 765 Pro Pro Ser Thr Pro Pro Gly Pro Glu Pro AlaPro Ala Gln Pro Ala 770 775 780 Ala Pro Arg Ala Ala Ala Ala Gln Ala ArgPro Arg Pro Val Ala Val 785 790 795 800 Ser Arg Arg Pro Ala Glu Gly ProAsp Pro Leu Gly Gly Trp Arg Arg 805 810 815 Gln Pro Pro Gly Pro Ser HisThr Ala Ala Pro Ala Ala Ala Ala Leu 820 825 830 Glu Ala Tyr Cys Ser ProArg Ala Val Ala Glu Leu Thr Asp His Pro 835 840 845 Leu Phe Pro Val ProTrp Arg Pro Ala Leu Met Phe Asp Pro Arg Ala 850 855 860 Leu Ala Ser IleAla Ala Arg Cys Ala Gly Pro Ala Pro Ala Ala Gln 865 870 875 880 Ala AlaCys Gly Gly Gly Asp Asp Asp Asp Asn Pro His Pro His Gly 885 890 895 AlaAla Gly Gly Arg Leu Phe Gly Pro Leu Arg Ala Ser Gly Pro Leu 900 905 910Arg Arg Met Ala Ala Trp Met Arg Gln Ile Pro Asp Pro Glu Asp Val 915 920925 Arg Val Val Val Leu Tyr Ser Pro Leu Pro Gly Glu Asp Leu Ala Gly 930935 940 Gly Gly Ala Ser Gly Gly Pro Pro Glu Trp Ser Ala Glu Arg Gly Gly945 950 955 960 Leu Ser Cys Leu Leu Ala Ala Leu Ala Asn Arg Leu Cys GlyPro Asp 965 970 975 Thr Ala Ala Trp Ala Gly Asn Trp Thr Gly Ala Pro AspVal Ser Ala 980 985 990 Leu Gly Ala Gln Gly Val Leu Leu Leu Ser Thr ArgAsp Leu Ala Phe 995 1000 1005 Ala Gly Ala Val Glu Phe Leu Gly Leu LeuAla Ser Ala Gly Asp Arg 1010 1015 1020 Arg Leu Ile Val Val Asn Thr ValArg Ala Cys Asp Trp Pro Ala Asp 1025 1030 1035 1040 Gly Pro Ala Val SerArg Gln His Ala Tyr Leu Ala Cys Glu Leu Leu 1045 1050 1055 Pro Ala ValGln Cys Ala Val Arg Trp Pro Ala Ala Arg Asp Leu Arg 1060 1065 1070 ArgThr Val Leu Ala Ser Gly Arg Val Phe Gly Pro Gly Val Phe Ala 1075 10801085 Arg Val Glu Ala Ala His Ala Arg Leu Tyr Pro Asp Ala Pro Pro Leu1090 1095 1100 Arg Leu Cys Arg Gly Gly Asn Val Arg Tyr Arg Val Arg ThrArg Phe 1105 1110 1115 1120 Gly Pro Asp Thr Pro Val Pro Met Ser Pro ArgGlu Tyr Arg Arg Ala 1125 1130 1135 Val Leu Pro Ala Leu Asp Gly Arg AlaAla Ala Ser Gly Thr Thr Asp 1140 1145 1150 Ala Met Ala Pro Gly Ala ProAsp Phe Cys Glu Glu Glu Ala His Ser 1155 1160 1165 His Ala Ala Cys AlaArg Trp Gly Leu Gly Ala Pro Leu Arg Pro Val 1170 1175 1180 Tyr Val AlaLeu Gly Arg Glu Ala Val Arg Ala Gly Pro Ala Arg Trp 1185 1190 1195 1200Arg Gly Pro Arg Arg Asp Phe Cys Ala Arg Ala Leu Leu Glu Pro Asp 12051210 1215 Asp Asp Ala Pro Pro Leu Val Leu Arg Gly Asp Asp Asp Gly ProGly 1220 1225 1230 Ala Leu Pro Pro Ala Pro Pro Gly Ile Arg Trp Ala SerAla Thr Gly 1235 1240 1245 Arg Ser Gly Thr Val Leu Ala Ala Ala Gly AlaVal Glu Val Leu Gly 1250 1255 1260 Ala Glu Ala Gly Leu Ala Thr Pro ProArg Arg Glu Val Val Asp Trp 1265 1270 1275 1280 Glu Gly Ala Trp Asp GluAsp Asp Gly Gly Ala Phe Glu Gly Asp Gly 1285 1290 1295 Val Leu 1446 basepairs nucleic acid single linear unknown 3 ATGGCCTGTC GTAAGTTTTGTCGCGTTTAC GGGGGACAGG GCAGGAGGAA GGAGGAGGCC 60 GTCCCGCCGG AGACAAAGCCGTCCCGGGTG TTTCCTCATG GCCCCTTTTA TACCCCAGCC 120 GAGGACGCGT GCCTGGACTCCCCGCCCCCG GAGACCCCCA AACCTTCCCA CACCACACCA 180 CCCAGCGAGG CCGAGCGCCTGTGTCATCTG CAGGAGATCC TTGCCCAGAT GTACGGAAAC 240 CAGGACTACC CCATAGAGGACGACCCCAGC GCGGATGCCG CGGACGATGT CGACGAGGAC 300 GCCCCGGACG ACGTGGCCTATCCGGAGGAA TACGCAGAGG AGCTTTTTCT GCCCGGGGAC 360 GCGACCGGTC CCCTTATCGGGGCCAACGAC CACATCCCTC CCCCGTGTGG CGCATCTCCC 420 CCCGGTATAC GACGACGCAGCCGGGATGAG ATTGGGGCCA CGGGATTTAC CGCGGAAGAG 480 CTGGACGCCA TGGACAGGGAGGCGGCTCGA GCCATCAGCC GCGGCGGCAA GCCCCCCTCG 540 ACCATGGCCA AGCTGGTGACTGGCATGGGC TTTACGATCC ACGGAGCGCT CACCCCAGGA 600 TCGGAGGGGT GTGTCTTTGACAGCAGCCAT CCAGATTACC CCCAACGGGT AATCGTGAAG 660 GCGGGGTGGT ACACGAGCACGAGCCACGAG GCGCGACTGC TGAGGCGACT GGACCACCCG 720 GCGATCCTGC CCCTCCTGGACCTGCATGTC GTCTCCGGGG TCACGTGTCT GGTCCTCCCC 780 AAGTACCAGG CCGACCTGTATACCTATCTG AGTAGGCGCC TGAACCCACT GGGACGCCCG 840 CAGATCGCAG CGGTCTCCCGGCAGCTCCTA AGCGCCGTTG ACTACATTCA CCGCCAGGGC 900 ATTATCCACC GCGACATTAAGACCGAAAAT ATTTTTATTA ACACCCCCGA GGACATTTGC 960 CTGGGGGACT TTGGCGCCGCGTGCTTCGTG CAGGGTTCCC GATCAAGCCC CTTCCCCTAC 1020 GGAATCGCCG GAACCATCGACACCAACGCC CCCGAGGTCC TGGCCGGGGA TCCGTATACC 1080 ACGACCGTCG ACATTTGGAGCGCCGGTCTG GTGATCTTCG AGACTGCCGT CCACAACGCG 1140 TCCTTGTTCT CGGCCCCCCGCGGCCCCAAA AGGGGCCCGT GCGACAGTCA GATCACCCGC 1200 ATCATCCGAC AGGCCCAGGTCCACGTTGAC GAGTTTTCCC CGCATCCAGA ATCGCGCCTC 1260 ACCTCGCGCT ACCGCTCCCGCGCGGCCGGG AACAATCGCC CGCCGTACAC CCGACCGGCC 1320 TGGACCCGCT ACTACAAGATGGACATAGAC GTCGAATATC TGGTTTGCAA AGCCCTCACC 1380 TTCGACGGCG CGCTTCGCCCCAGCGCCGCA GAGCTGCTTT GTTTGCCGCT GTTTCAACAG 1440 AAATGA 1446 481 aminoacids amino acid linear unknown 4 Met Ala Cys Arg Lys Phe Cys Arg ValTyr Gly Gly Gln Gly Arg Arg 1 5 10 15 Lys Glu Glu Ala Val Pro Pro GluThr Lys Pro Ser Arg Val Phe Pro 20 25 30 His Gly Pro Phe Tyr Thr Pro AlaGlu Asp Ala Cys Leu Asp Ser Pro 35 40 45 Pro Pro Glu Thr Pro Lys Pro SerHis Thr Thr Pro Pro Ser Glu Ala 50 55 60 Glu Arg Leu Cys His Leu Gln GluIle Leu Ala Gln Met Tyr Gly Asn 65 70 75 80 Gln Asp Tyr Pro Ile Glu AspAsp Pro Ser Ala Asp Ala Ala Asp Asp 85 90 95 Val Asp Glu Asp Ala Pro AspAsp Val Ala Tyr Pro Glu Glu Tyr Ala 100 105 110 Glu Glu Leu Phe Leu ProGly Asp Ala Thr Gly Pro Leu Ile Gly Ala 115 120 125 Asn Asp His Ile ProPro Pro Cys Gly Ala Ser Pro Pro Gly Ile Arg 130 135 140 Arg Arg Ser ArgAsp Glu Ile Gly Ala Thr Gly Phe Thr Ala Glu Glu 145 150 155 160 Leu AspAla Met Asp Arg Glu Ala Ala Arg Ala Ile Ser Arg Gly Gly 165 170 175 LysPro Pro Ser Thr Met Ala Lys Leu Val Thr Gly Met Gly Phe Thr 180 185 190Ile His Gly Ala Leu Thr Pro Gly Ser Glu Gly Cys Val Phe Asp Ser 195 200205 Ser His Pro Asp Tyr Pro Gln Arg Val Ile Val Lys Ala Gly Trp Tyr 210215 220 Thr Ser Thr Ser His Glu Ala Arg Leu Leu Arg Arg Leu Asp His Pro225 230 235 240 Ala Ile Leu Pro Leu Leu Asp Leu His Val Val Ser Gly ValThr Cys 245 250 255 Leu Val Leu Pro Lys Tyr Gln Ala Asp Leu Tyr Thr TyrLeu Ser Arg 260 265 270 Arg Leu Asn Pro Leu Gly Arg Pro Gln Ile Ala AlaVal Ser Arg Gln 275 280 285 Leu Leu Ser Ala Val Asp Tyr Ile His Arg GlnGly Ile Ile His Arg 290 295 300 Asp Ile Lys Thr Glu Asn Ile Phe Ile AsnThr Pro Glu Asp Ile Cys 305 310 315 320 Leu Gly Asp Phe Gly Ala Ala CysPhe Val Gln Gly Ser Arg Ser Ser 325 330 335 Pro Phe Pro Tyr Gly Ile AlaGly Thr Ile Asp Thr Asn Ala Pro Glu 340 345 350 Val Leu Ala Gly Asp ProTyr Thr Thr Thr Val Asp Ile Trp Ser Ala 355 360 365 Gly Leu Val Ile PheGlu Thr Ala Val His Asn Ala Ser Leu Phe Ser 370 375 380 Ala Pro Arg GlyPro Lys Arg Gly Pro Cys Asp Ser Gln Ile Thr Arg 385 390 395 400 Ile IleArg Gln Ala Gln Val His Val Asp Glu Phe Ser Pro His Pro 405 410 415 GluSer Arg Leu Thr Ser Arg Tyr Arg Ser Arg Ala Ala Gly Asn Asn 420 425 430Arg Pro Pro Tyr Thr Arg Pro Ala Trp Thr Arg Tyr Tyr Lys Met Asp 435 440445 Ile Asp Val Glu Tyr Leu Val Cys Lys Ala Leu Thr Phe Asp Gly Ala 450455 460 Leu Arg Pro Ser Ala Ala Glu Leu Leu Cys Leu Pro Leu Phe Gln Gln465 470 475 480 Lys 10 amino acids amino acid linear unknown 5 Arg ArgArg Arg Thr Arg Arg Ser Arg Glu 1 5 10 6 amino acids amino acid linearunknown Modified-site /note= “Residue may be arginine or any amino acidresidue.” Modified-site /note= “Residue may be either serine orthreonine.” Modified-site /note= “Residue may be either arginine ortyrosine.” 6 Arg Arg Arg Arg Ser Arg 1 5

What is claimed is:
 1. A screening method for compounds havinginhibitory activity against herpes simplex virus U_(s)3polypeptide-induced inhibition of apoptosis comprising the steps of: (a)providing a first cell comprising a herpes simplex virus U_(s)3 geneunder the control of a herpes simplex virus immediate early promoter;(b) infecting said first cell with a herpes simplex virus that lacks afunctional U_(s)3 gene; (c) contacting said first cell with a testcompound; (d) incubating said first cell under conditions permittingviral replication; and (e) comparing the cell pathology of said firstcell following incubation with (i) the cell pathology of a second cellthat lacks a herpes simplex virus U_(s)3 gene following infection withsaid herpes simplex virus and contact with said test compound and (ii)the cell pathology of a third cell comprising a herpes simplex virusU_(s)3 gene under the control of a herpes simplex virus immediate earlypromoter following infection with said herpes simplex virus but in theabsence of said test compound.
 2. The method of claim 1, wherein saidcell pathology comprises condensation of chromatin, obliteration ofnuclear membranes, vacuolization, cytoplasmic blebbing, and DNAfragmentation.
 3. The method of claim 1, wherein said first cellcontains an integrated copy of a wild-type herpes simplex virus geneunder the control of an U_(s)3 promoter.
 4. The method of claim 3,wherein said herpes simplex virus that lacks a functional herpes simplexvirus has a deletion in both copies of the virally-encoded U_(s)3 genes.5. The method of claim 4, wherein said herpes simplex virus that lacks afunctional herpes simplex virus U_(s)3 gene carries a temperaturesensitive mutation in both copies of the virally-encoded U_(s)3 gene andthe incubation is at 39.5° C.
 6. A screening method for compounds havinginhibitory activity against herpes simplex virus U_(s)3-inducedinhibition of apoptosis comprising the steps of: (a) providing a firstcell comprising a herpes simplex virus U_(s)3 gene under the control ofan inducible promoter; (b) inducing said transcription from saidpromoter; (c) contacting said first cell with a test compound; (d)incubating said first cell under conditions permitting expression of aherpes simplex virus U_(s)3 polypeptide; and (e) comparing the cellpathology of said first cell following incubation with (i) the cellpathology of a second cell not having a herpes simplex virus U_(s)3 genefollowing induction and (ii) the cell pathology of a third cellcomprising a herpes simplex virus U_(s)3 gene under the control of aninducible promoter following induction but in the absence of said testcompound.
 7. A screening method for compounds having inhibitory activityagainst herpes simplex virus U_(s)3 polypeptide-induced inhibition ofapoptosis comprising the steps of: (a) providing a first cell; (b)infecting said cell with a herpes simplex virus comprising a U_(s)3gene; (c) contacting said first cell with a test compound; (d)incubating said first cell under condition permitting viral replication;and (e) comparing the cell pathology of said first cell followingincubation with (i) the cell pathology of a second cell treated withsaid test compound alone and (ii) the cell pathology of a third cellfollowing infection with said herpes simplex virus but in the absence ofsaid test compound.
 8. The method of claim 1, wherein said contacting ofsaid first cell with a test compound occurs directly.
 9. The method ofclaim 1, wherein said test compound is formulated to provide improvedcellular uptake.
 10. The method of claim 6, wherein said cell pathologycomprises condensation of chromatin, obliteration of nuclear membranes,vacuolization, cytoplasmic blebbing and DNA fragmentation.
 11. Themethod of claim 6, wherein said first cell contains an integrated copyof a wild-type herpes simplex virus gene.
 12. The method of claim 6,wherein said contacting of said first cell with a test compound occursdirectly.
 13. The method of claim 6, wherein said test compound isformulated to provide improved cellular uptake.
 14. The method of claim7, wherein said cell pathology comprises condensation of chromatin,obliteration of nuclear membranes, vacuolization, cytoplasmic blebbingand DNA fragmentation.
 15. The method of claim 7, wherein said U_(s)3gene is under the control of an U_(s)3 promoter.
 16. The method of claim7, wherein contacting said first cell with a test compound occurs priorto infecting said cell with a herpes simplex virus comprising a U_(s)3gene.
 17. The method of claim 7, wherein contacting said first cell witha test compound occurs at the same time as infecting said cell with aherpes simplex virus comprising a U_(s)3 gene.
 18. The method of claim7, wherein contacting said first cell with a test compound occurs afterinfecting said cell with a herpes simplex virus comprising a U_(s)3gene.
 19. The method of claim 7, wherein said contacting of said firstcell with a test compound occurs directly.
 20. The method of claim 7,wherein said test compound is formulated to provide improved cellularuptake.